Mechanism of SETX-BRCA1-BARD1 complex in resolution of R-loops and transcription-replication conflicts

Mechanism of SETX-BRCA1-BARD1 complex in resolution of R-loops and transcription-replication conflicts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mechanism of SETX-BRCA1-BARD1 complex in resolution of R-loops and transcription-replication conflicts Patrick Sung, Arijit Dutta, Jae-Hoon Ji, Qingming Fang, Shuo Zhou, and 22 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3833044/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Mar, 2026 Read the published version in Nature Structural & Molecular Biology → Version 1 posted You are reading this latest preprint version Abstract Senataxin (SETX), a putative RNA-DNA helicase, is recruited to transcription pause sites via the tumor suppressor BRCA1. Here, we define the mechanism by which SETX-BRCA1 resolves transcription-associated R-loops to prevent deleterious outcomes. Specifically, we show that SETX unwinds R-loops, and that the complex of BRCA1 and its obligatory partner BARD1 binds R-loops and stimulates R-loop unwinding by SETX. Importantly, BRCA1-BARD1 alleviates the inhibitory effect of RAD52 on SETX-mediated R-loop unwinding. We also demonstrate that phosphorylation of Ser642 in SETX promotes its interaction with BRCA1 via the tandem BRCT domain of the latter. Accordingly, mutations in the catalytic domain or Ser642 in SETX lead to R-loop accumulation, transcription-replication conflicts, replication fork stalling, and DNA double strand breaks in human cells. Our results thus establish the molecular basis for functional synergy between SETX and BRCA1-BARD1 in R-loop resolution and the mitigation of transcription-replication conflicts to preserve genome integrity. Biological sciences/Biochemistry Biological sciences/Cell biology R-loop SETX BRCA1-BARD1 phospho-SPTF motif replication-transcription conflict Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction R-loops are three-stranded nucleic acid structures that harbor a RNA transcript hybridized to a DNA template and serve important physiological roles, including gene expression regulation and Ig class-switch recombination 1,2 . However, excessive R-loops can compromise genome stability as they impede the progression of replication forks in dividing cells and can lead to the induction of DNA double strand breaks (DSBs) via nucleolytic cleavage of the ssDNA within the R-loop structure 2–4 . Senataxin (SETX), a putative RNA-DNA helicase that promotes transcription termination 5 , was identified as a BRCA1 interactor in a proteomic analysis 6 . Subsequent studies have implicated the SETX-BRCA1 complex in the maintenance of genomic stability at transcribing genomic regions via R-loop removal 7 . SETX accumulates at DSBs in a transcription-dependent manner, and provides resistance against oxidative stress and genotoxic agents such as camptothecin (CPT) and mitomycin C (MMC) 8,9 . SETX plays a role in the homology-directed repair of DSBs 10 . Depletion of SETX or BRCA1 also leads to increased accumulation of cytosolic RNA-DNA hybrids and activation of innate immune signaling via cGAS-STING pathway due to XPG-mediated processing of nuclear R-loop 11 . Hereditary mutations in SETX are associated with two motor neuron diseases – a rare juvenile form of amyotrophic lateral sclerosis (ALS4) 12 , and ataxia oculomotor apraxia type 2 (AOA2) 13 , where pathogenicity has been largely associated with dysregulation of R-loop homeostasis in neurons 14 . SETX is a very large protein of 2,677 amino acid residues (303 kDa). It possesses within its C-terminal region (residues 1736–2464) a domain conserved within the Superfamily 1B (SF1B) helicases. It also harbors a large region (residues 596–1735) predicted to be disordered (Fig. 1a). The helicase core domain of SETX is structurally homologous to yeast Sen1 15 , which can unwind RNA-DNA a 5′ to 3′ direction with regards to the RNA strand 16–18 , to facilitate transcription termination 18–20 , maintain genome stability via resolution of RNA-DNA hybrids at replication forks, and prevent transcription-replication conflicts 21,22 . To elucidate the R-loop resolution mechanism of human SETX and its possible functional synergy with BRCA1, we have developed systems for expressing and purifying full length human SETX to near homogeneity (Fig. 1b) for biochemical testing. We demonstrate that SETX possesses a helicase activity that efficiently unwinds R-loops, and that the breast cancer associated mutations N2010I and H2197R impair SETX activity (Fig. 1d,e). Importantly, we also present data to show that SETX physically interacts with BRCA1 through the tandem BRCT domain of the latter and that SETX-BRCA1 complex formation is enhanced by the phosphorylation of S642 in SETX within the context of a SPTF motif. Our in vitro studies demonstrate BRCA1 in complex with its obligatory partner BARD1 binds R-loops and stimulates SETX-catalyzed R-loop dissociation that is dependent on the interaction between SETX and BRCA1. On the contrary, SETX-mediated R-loop dissociation is impeded by RAD52, a homologous recombination factor that has been implicated in both BRCA1-dependent 23 and BRCA1-independent transcription associated DSB repair processes 24 . Importantly, this inhibitory effect of RAD52 is efficiently overcome by BRCA1-BARD1. We further demonstrate that human cells expressing SETX-catalytic mutants and the BRCA1 interaction defective S642A mutant accumulate R-loops and DSBs, and also exhibit hypersensitivity to Olaparib, an inhibitor of polyADP ribose polymerases (PARPs). Interestingly, increased RAD52 foci in cells expressing SETX mutants suggest that R-loop associated DSBs undergo repair via a RAD52-dependent mechanism in such a cellular setting. Finally, we show that SETX is recruited to stressed replication forks in a BRCA1-depdendent manner and is indispensable for replication fork progression. Altogether, our findings shed light on the molecular mechanism of the SETX-BRCA1-BARD1 axis in R-loop resolution and replication fork maintenance. Results Nucleic acid unwinding activities of SETX SETX harbors Walker A and Walker B motifs associated with ATP binding and hydrolysis (Fig. 1 a) 25 . To obtain human SETX, we constructed a recombinant baculovirus to express the full-length protein with N-terminal 6X-His-MBP and C-terminal FLAG tags in insect cells. SETX thus expressed is soluble and can be purified to near homogeneity (Fig. 1 b). Purified SETX hydrolyzes ATP with a strong dependence on either RNA or DNA, but shows no preference for either nucleic acid species with a K cat of ~ 180 min − 1 (Fig. 1 c). We asked whether SETX binds R-loops using the electrophoretic mobility shift assay (EMSA) with two different R-loop substrates having either a 5′-RNA overhang or no overhang. SETX bound these R-loops with a comparable affinity (Extended Data Fig. 1 a). We generated two SETX mutants, SETX-K1969A (KA) and SETX-E2182A (EA), by mutating the highly conserved Lys1969 in the Walker A motif and Glu2182 in the Walker-B motif to Alanine (Fig. 1 a,b, Extended Data Fig. 1 b,c). As expected, both the SETX-KA and SETX-EA mutants failed to hydrolyze ATP (Fig. 1 d). Next, we searched for pathological SETX mutations in the cBioPortal 26 , a cancer somatic mutation database, particularly focusing on the mutations occurring within the helicase domain, and chose two metastatic breast cancer-associated mutations N2010I (NI) and H2197R (HR) for mechanistic characterization. N2010 is located in motif Ia of SETX that is likely involved in nucleic acid binding, and His2197 is located between motif II/Walker-B and motif III likely required for ATP binding and hydrolysis (Fig. 1 a, Extended Data Fig. 1 b,c). Interestingly, the N2010S and H2197R SETX mutations are associated with the neurodegenerative disease ataxia with oculomotor apraxia type 2 27,28 , indicating that these genetic alterations have pathogenic consequences. SETX-NI and SETX-HR mutant proteins were purified from insect cells to near homogeneity following the same purification scheme as SETX-WT (Fig. 1 b). The results showed that both pathogenic mutations engender a defect in ATP hydrolysis (Fig. 1 d). Next, we tested SETX activity on R-loops generated with oligonucleotides that harbor a 5′ or 3′ RNA overhang or without any such overhang (Supplementary Tables 1 and 2). SETX unwound all three R-loop substrates when ATP was present (Fig. 1 e, Extended Data Fig. 2 a) but not when ATP was substituted with the nonhydrolyzable ATP analog AMP-PNP (Extended Data Fig. 1 d, lanes # 6, 12), indicating that R-loop unwinding is driven by ATP hydrolysis. SETX activity was the strongest on the R-loop substrate with a 5′-RNA overhang, followed by the substrate with a 3′-RNA overhang, then that with no overhang (Fig. 1 e, Extended Data Fig. 2 a). Importantly, the Walker-domain mutants SETX-KA and SETX-EA and pathogenic mutants SETX-NI and SETX-HR all displayed defects in R-loop unwinding (Fig. 1 e). To further define the substrate specificity of SETX helicase activity, we also tested duplex DNA, RNA-DNA, and R-loop substrates with a 30-nt 5′ or 3′ single stranded (ss) DNA or RNA overhangs (Extended Data Fig. 2 a,b). Importantly, the results showed that SETX also unwinds D-loop (Extended Data Fig. 2 a, lane # 26–30) and dsDNA with either a 5′ or 3′ overhang (Extended Data Fig. 2 b, lane # 1–10), while it cannot unwind blunt-ended dsDNA (Extended Data Fig. 2 C). Overall, the results indicated that SETX is more proficient at unwinding R-loops than any of the DNA-based substrates (Extended Data Fig. 2 A). Moreover, SETX has only modest activity toward RNA-DNA substrates with either a 3′ or 5′ DNA or RNA overhang (Extended Data Fig. 2 B, lane # 11–30), while it is devoid of the ability to unwind dsRNA with a 5′ RNA overhang (Extended Data Fig. 2 d). We note that SETX is just as proficient in dissociating substrates that resemble R-loops but without the displaced ssDNA strand (Extended Data Fig. 2 a, lane # 16–25). Altogether, our results indicate that SETX unwinds RNA-DNA hybrids embedded within dsDNA robustly and without any requirement for a displaced ssDNA strand. SETX activity on R-loops embedded within a plasmid Having established that SETX is adept at dissociating synthetic R-loops constructed with oligonucleotides, we wished to determine whether SETX can also act on more physiologically relevant R-loop intermediates formed during transcription. For this, we generated 32 P -labeled R-loops via in vitro transcription using T7 RNA polymerase. The template is embedded within a closed-circular dsDNA template containing ~ 1 kb telomeric repeat sequence, referred to as pTelo (Fig. 1 f, schematics) 29 . Importantly, we found that SETX unwinds R-loops generated within the telomeric sequence robustly with a strict dependence on ATP hydrolysis (Fig. 1 f), but that all four SETX mutants, namely, K1969A, E2182A, N2010I and H2197R, are defective in this regard (Extended Data Fig. 1 e). Dissection of SETX-BRCA1 interaction SETX was identified as a BRCA1-interacting factor in a proteomic analysis 6 . These two proteins co-immunoprecipitate from lysates of human cells and it has been suggested that BRCA1 and SETX function together in maintaining genome stability at transcription pause sites via R-loop resolution 7 . Other studies have also implicated BRCA1 in mitigating R-loop associated genome instability, which is linked with tumorigenicity 30,31 . Our own immunoprecipitation analysis using HEK293 cell lysate recapitulated co-precipitation of SETX with the BRCA1-BARD1 complex. The amount of coprecipitating SETX-BRCA1-BARD1 increased upon treatment of cells with CPT, a topoisomerase 1 (TOP1) inhibitor, that is known to enhance R-loop accumulation via generation of TOP1 cleavage complex and RNA Polymerase II (RNA Pol II) pausing (Fig. 2 a) 32 . Next, we addressed whether SETX interacts with BRCA1-BARD1 directly. For this, we followed our published procedures to co-express BRCA1 and BARD1 in insects and purify the assembled complex to near homogeneity 33 (Extended Data Fig. 3 a). Importantly, affinity pulldown analysis done in the presence of benzonase (a pan nuclease that digests both DNA and RNA) provided evidence that SETX directly interacts with BRCA1-BARD1 (Fig. 2 b). Previously, SETX was reported to co-immunoprecipitate with a BRCA1 fragment (amino acid residues 1568–1863) that encompasses the tandem BRCT (tBRCT) domain of BRCA1 7 . To ask whether the BRCA1 tBRCT domain is required for SETX interaction, we generated BRCA1 ΔBRCT -BARD1 that lacks the BRCA1 tBRCT domain (Extended Data Fig. 3 a). Our pulldown result showed that BRCA1 ΔBRCT -BARD1 is indeed defective in SETX interaction (Fig. 2 c). To map the region in SETX that interacts with BRCA1-BRCT, we generated SETX-HD (amino acid residues 1932–2677) and SETX ΔHD (amino acid residues 1-1721) (Extended Data Fig. 3 b) for pulldown analysis. we found that SETX ΔHD interacts with BRCA1-BARD1 with an affinity comparable to full length SETX (Fig. 2 d, lanes # 3–6). Importantly, SETX ΔHD failed to interact with BRCA1 ΔBRCT -BARD1 (Fig. 2 d, lanes # 7–10), but showed strong interaction with BRCA1-tBRCT (Fig. 2 e, lanes # 3–4). However, SETX ΔHD exhibited little or no affinity toward the BARD1 tBRCT domain (Fig. 2 e, lanes # 5–6), which aptly underscores the specificity of the SETX-BRCA1 tBRCT interaction. Finally, we observed that SETX-HD also interacts with the BRCA1-tBRCT domain, albeit only weakly (Fig. 2 f, lane # 6). Altogether, our domain mapping effort provided evidence that SETX forms a complex with BRCA1-BARD1 via two interaction regions, one located within the N-terminal half of the protein, and other within its helicase domain. Role of SETX-Ser642 phosphorylation in SETX-BRCA1 interaction. A number of BRCA1 interacting factors, such as BACH1/FANCJ, Abraxas, and CtIP, relies on a consensus SPTF motif, in which the Ser residue is subject to phosphorylation, for complex formation with the tBRCT domain of BRCA1 34–37 . Importantly, SETX possesses a S 642 PTF motif that is highly conserved in mammals but not in lower metazoans (Fig. 3 a,b). Phosphorylation of Ser642 in SETX peptides was reported in multiple whole cell phospho-proteomic studies 38,39 . Our own mass-spectrometric analysis of FLAG-affinity purified SETX expressed in Expi293F cells (Extended Data Fig. 4 a-c), revealed that SETX-Ser642 is phosphorylated constitutively, with the extent of phosphorylation increased upon treatment of cells with CPT (Supplementary Tables 3, 4). Moreover, with a custom rabbit polyclonal antibody (Biomatik) against the SETX phospho-peptide CLEAS[ pS ]PTFSKEPM, we showed that CPT treatment of insect cells led to an elevation of Ser642 phosphorylation level in recombinant SETX as well (Extended Data Fig. 4 d). Next, we examined the association of a carboxyfluorescein (FAM)-labelled SETX phospho-SPTF peptide with BRCA1-BARD1 and the tBRCT domain from either BRCA1 or BARD1 (Extended Data Fig. 5 a,b). Specifically, we incubated the labeled SETX peptide with either FLAG-tagged BRCA1-BARD1, or GST-tagged BRCA1-tBRCT and BARD1-tBRCT, followed by affinity pulldown via the affinity tags and analyzed eluates from the affinity matrices by dot blot or immunoblot analysis (Extended Data Fig. 5 a,b). We found that the SETX phospho-peptide interacts with full-length BRCA1-BARD1 and BRCA1-tBRCT, but not BARD1-tBRCT. We also employed microscale thermophoresis (MST) to independently verify conclusions drawn from the affinity pulldown experiments. The results confirmed that the phospho-SPTF peptide possesses a much stronger affinity (K d [95% CI] = 206 nM) toward the BRCA1-tBRCT domain (Fig. 3 c) than does the non-phosphorylated counterpart of this peptide (Extended Data Fig. 5 c). Interestingly, the phospho-mimetic EPTF peptide bound BRCA1-tBRCT with ~ 30-fold weaker affinity, while DPTF peptide did not exhibit significant binding affinity, suggesting these mutations do not truly mimic phospho-Ser. As expected, peptides with Ser642 or Phe645 replaced with Ala also did not show significant affinity for BRCA1-tBRCT (Extended Data Fig. 5 c). Collectively, the results presented provide evidence that phosphorylation of SETX-Ser642 facilitates the formation of the SETX-BRCA1 complex. Finally, to further corroborate the requirement of SETX-pSer642 for BRCA1-interaction, we generated SETX-S642A (SA) constructs for expression in insect cells and human cells. FLAG-co-IP from 293T cells co-expressing GFP, FLAG-tagged SETX-WT or SETX-SA, and Myc-tagged BRCA1 showed a significantly reduced interaction of SETX-SA with BRCA1 (Fig. 3 d). The breast cancer associated BRCA1 pathogenic variants S1665F (SF) and M1775R (MR) were reported to destablize the tBRCT domain and disrupt its interaction with the SxxF motif of BACH1/FANCJ, Abraxas, and CtIP 34–37 . In BACH1/FANCJ, phospho-Ser990 of the phospho-SxxF motif forms hydrogen bonds with Ser1655 and Lys1702 of BRCA1, while the Phe993 residue fits in a hydrophobic pocket comprised of Phe1704, Met1775 and Leu1839 of BRCA1 36 . We co-expressed Myc-tagged BRCA1-WT, and the mutants, ΔBRCT, SF or MR with GFP-FLAG-tagged SETX in 293T cells and performed co-IP studies, which showed that the BRCA1 mutants are impaired for SETX interaction (Fig. 3 e). Enhancement of SETX activity by BRCA1-BARD1 Having established direct interaction between SETX and BRCA1-BARD1, we next investigated possible functional synergy of these protein factors in R-loop resolution. Firstly, we examined BRCA1-BARD1 binding to R-loops in vitro . Both BRCA1-BARD1 and BRCA1 ΔBRCT -BARD1 bound R-loops with comparable affinities (Fig. 4 a), indicating that deletion of the BRCA1-tBRCT domain does not significantly impact the R-loop binding activity of BRCA1-BARD1. However, SETX-mediated unwinding of telo R-loop was stimulated by BRCA1-BARD1, but not by BRCA1 ΔBRCT -BARD1 that does not interact with SETX (Fig. 4 b). We note that BRCA1-BARD1, but not BRCA1 ΔBRCT -BARD1 could also stimulate unwinding of short synthetic R-loop via SETX (Extended Data Fig. 2 e). Next, we asked whether the RAD52 protein (Extended Data Fig. 3 c), which binds R-loops avidly (Extended Data Fig. 3 d) and physically interacts with SETX in vitro and in cells (Extended Data Fig. 3 e,f), would affect the R-loop resolution activity of SETX. Importantly, we found that RAD52, at amounts stoichiometric to that of SETX, strongly inhibits R-loop resolution by SETX (Fig. 4 C). In this analysis, we also included the human RAD52 K144A mutant protein that is impaired for R-loop binding (Extended Data Fig. 3 c,d) as previously reported 24 . The results showed that RAD52 K144A failed to inhibit SETX mediated R-loop unwinding, thus providing direct evidence to link R-loop binding by RAD52 to its inhibitory action on SETX-mediated R-loop resolution. Importantly, we found that BRCA1-BARD1 helps alleviate the inhibitory effect of RAD52 on R-loop unwinding by SETX (Fig. 4 d). These results highlight stimulatory effects of BRCA1-BARD1 on SETX’s R-loop unwinding activity and a potential mechanism for overcoming the inhibitory effects of R-loop binding factors such as RAD52. Requirement of SETX S642 phosphorylation for R-loop resolution in cells We next carried out cell-based studies to interrogate the physiological significance of the SETX-BRCA1-BARD1 axis of R-loop resolution. Firstly, we observed that HAP1 SETX-KO cells, as compared to isogenic wild type cells, are hypersensitive to camptothecin (CPT) treatment (Extended Data Fig. 6 a,b) and accumulate R-loops as revealed by immunostaining with the S9.6 antibody (Extended Data Fig. 6 c), which recognizes RNA-DNA hybrids, and by S9.6 slot blot analysis (Extended Data Fig. 6 d). Importantly, we confirmed that treatment of RNase H1, but not with RNase III, RNase T1, and RNase A, reduces DNA:RNA hybrids detection by S9.6 antibody (Extended Data Fig. 6 e). We also carried out SETX-S9.6 proximity ligation assay (PLA) to test whether SETX associates with R-loops in cells. The results revealed that SETX colocalizes with R-loops in the nucleolus and nucleoplasm, whereas the treatment of RNase H1, but not with other RNases, disrupts the interaction between SETX and R-loops (Extended Data Fig. 6 f). Moreover, CPT treatment dramatically enhances the association of SETX with R-loops (Extended Data Fig. 6 g). To understand the significance of SETX’s catalytic activity and Ser642 phosphorylation in R-loop resolution, we constructed HEK293A cell lines stably expressing GFP and FLAG-tagged SETX-WT, EA (catalytic null) and SA (Ser642 phosphorylation null) mutants for testing. PLA analysis showed that while both SETX-WT and SETX-EA colocalized with R-loops, SETX-SA failed to do so, indicative of a requirement of Ser642 phosphorylation for SETX recruitment to R-loops (Fig. 5 a). Next, we performed S9.6-slot blot analysis of cells after down-regulation of endogenous SETX via a 3′-UTR targeting siRNA (Extended Data Fig. 6 h). While the ectopic expression of SETX-WT suppressed R-loop accumulation, cells expressing the SETX EA or SA mutant exhibited R-loop levels comparable to SETX-depleted cells that harbored the empty vector (Fig. 5 b, Extended Data Fig. 6 h). Treatment of SETX-depleted cells with and without Olaparib, an inhibitor of polyADP ribose polymerases (PARPi), also significantly increased γH2AX foci formation (Fig. 5 c,d), suggesting SETX is needed for the proper response to increased replication stress and avoidance of DNA double-strand breaks (DSBs) upon occurrence of such stress. Importantly, while expression of SETX-WT in these cells attenuated the level of γH2AX foci, neither the SETX-EA nor SETX-SA mutant was able to do so (Fig. 5 c,d), indicating that SETX catalytic activity and phospho-Ser642 are both important for preventing DSB formation linked to R-loop accumulation. Interestingly, we also found increased RAD52 foci in SETX depleted cells or when the SETX-EA or SETX-SA mutant was expressed in these cells (Fig. 5 e), suggesting that cells become more reliant on the RAD52-dependent repair mechanism 23,24 under these circumstances. SETX knockdown also reduced clonogenic survival of 293A cells upon olaparib treatment, and this phenotype could be corrected by ectopic expression of SETX-WT, but neither SETX-EA nor SETX-SA was effective in this regard (Fig. 5 f). SETX recruitment and function at replication forks The increased PARPi sensitivity of SETX depleted or mutant expressing cells suggests that SETX is required for protection of stressed replication forks. We used iPOND (isolation of proteins on nascent DNA) to examine possible recruitment of SETX to replication forks in HeLa cells. The results showed that association of SETX with replication forks becomes greatly enhanced upon treatment of cells with pladienolide-B (PB), an RNA splicing inhibitor that is known to induce R-loops (Fig. 6 a, lane # 11–12). As expected, we found that expression of RNase H1, which digests RNA in RNA-DNA hybrids, leads to attenuation of SETX recruitment to replication forks in PB-treated cells (Fig. 6 a, lane # 13–14). PB treatment also increased the level of γH2AX in replicating DNA that could be mitigated by RNaseH1 expression (Fig. 6 a, lane # 11–14). Next, we conducted iPOND analysis of cells in which BRCA1 could be depleted by doxycycline-inducible expression of shRNA against BRCA1 40 , which showed clearly that the recruitment of SETX to replication forks are dependent on BRCA1 (Fig. 6 a, lane # 15–18). We also utilized in situ analysis protein interaction at DNA replication forks (SIRF) 41 to show that SETX recruited to replicating DNA (Fig. 6 b). While GFP-tagged SETX-WT and SETX-EA formed SIRF foci with EdU incorporated nascent DNA in S-phase cells, fewer SETX-SA foci were seen in these cells, indicating that Ser642 phosphorylation is important for SETX recruitment and/or retention at replication forks (Fig. 6 b). Furthermore, we examined the effect of SETX depletion with or without co-expression of SETX mutants on transcription-replication conflicts in cells via PLA of proliferating cell nuclear antigen (PCNA) and RNA polymerase II (RPII), following the rationale given in other studies 42,43 . Specifically, it has been shown that transcriptionally active RP II associates with nascent DNA via a transient interaction with PCNA, which enables the resumption of transcription upon passing of the DNA replication machinery 44 . Depletion of SETX increased PCNA-RPII foci that could be eliminated via treatment with RNase H1 (Fig. 6 c). Importantly, SETX-EA or SETX-SA expressing cells harbored a significantly larger number of PCNA-RPII foci compared to those expressing SETX-WT (Fig. 6 c). Furthermore, we examined the effect of SETX depletion or mutations on DNA replication with SIRF assay that measured PCNA-EdU foci. SETX depletion led to reduced PCNA-EdU SIRF foci, which phenocopied the treatment of wild type cells with the replicative stressor hydroxyurea (HU) (Fig. 6 d). Importantly, expression of SETX-WT, but not either SETX-EA or SETX-SA in cells depleted for endogenous SETX restored PCNA-EdU SIRF foci (Fig. 6 d). Altogether, the data presented herein reveal that ( 1 ) R-loop accumulation leads to enhanced association of SETX with DNA replication forks, ( 2 ) recruitment of SETX to replication forks is dependent on its interaction with BRCA1 but its catalytic activity is dispensable, ( 3 ) SETX is needed for the avoidance of replication-transcription conflicts, and ( 4 ) SETX deficiency negatively impacts the progression of DNA replication forks, a premise that is supported by DNA fiber analysis as we present below. SETX is required for progression of replication forks We interrogated how SETX depletion and SETX mutations affect DNA replication by DNA fiber analysis. SETX knockdown led to a significant decrease in the IdU/CIdU ratio (Fig. 7 a,b) and an increase in the ratio of stalled forks, indicating that SETX is needed for fork progression (Fig. 7 c). In contrast, normal fork progression is restored in cells depleted for SETX but simultaneously expressing RNaseH1, providing evidence that fork stalling is a direct consequence of R-loop accumulation in the SETX deficient cells. Notably, ectopic expression of SETX-WT, but not SETX-EA or SETX-SA, fully alleviated the fork stalling phenotype induced by SETX ablation. We also wished to define the role of SETX in mitigating the effect of CPT-induced R-loops on DNA replication. For this purpose, we treated cells with 1 µM CPT for 30 min after CIdU labelling, followed by IdU labelling, and then performed DNA fiber analysis. As expected, fork progression (IdU/CIdU ratio in Fig. 7 d,e) and normal fork restart events (Fig. 7 f) were reduced in SETX knockdown cells. This aberrant phenotype could be overcome by expression of RNaseH1 or SETX-WT, but not by SETX-EA or SETX-SA. DISCUSSION Cell-based investigations have implicated SETX in R-loop avoidance, but, thus far, the challenge of purifying SETX has precluded mechanistic dissection of its role in this crucial genome maintenance process. Importantly, we have succeeded in obtaining highly purified full-length human SETX via expression in insect cells, allowing us to demonstrate its R-loop and nucleic acid unwinding activities that likely reflect a direct role of SETX in R-loop resolution in vivo. Furthermore, our studies have provided the requisite platform for determining the mechanism by which BRCA1-BARD1 regulates SETX activity. Previous studies on Sen1 and the SETX helicase domain suggested that SETX unwinds R-loop via binding the 5′-end of RNA, as would be consistent with a role of SETX in transcription termination 16–18 . However, we have now shown that SETX binds and unwinds R-loop substrates without a 5′ ssRNA overhang (Fig. 1 e, Extended Data Fig. 1 d, 2 a). From our biochemical analysis, we conclude that SETX unwind R-loops and RNA embedded within dsDNA efficiently without any requirement for an RNA overhang or displaced ssDNA as is encountered in the R-loop structure. Moreover, we demonstrate that somatic SETX point mutations identified in metastatic breast cancer patients, namely N2010I (NI) and H2197R (HR), in the helicase domain, engender a dramatic defect in catalytic activities (Fig. 1 d,e, Extended Data Fig. 1 e), suggesting an etiological linkage of SETX R-loop unwinding defect with tumorigenesis. BRCA1 is recruited to transcription pause sites that have a high propensity of accumulating R-loops, where BRCA1 is thought to promote transcription termination via SETX 7 . Published studies have furnished evidence for a role of BRCA1 in R-loop resolution in specific genomic regions, and loss of BRCA1 lead to R-loop accumulation in breast tumors 30,45 . However, it has remained unclear whether BRCA1 directly interacts with SETX and if it participates in R-loop resolution with SETX. In this study, we have demonstrated a physical complex of SETX with BRCA1-BARD1 and that phosphorylation of the serine residue in the S 642 PTF motif of SETX enhances its interaction with the tBRCT domain of BRCA1. The SETX-S642A (SA) mutation and BRCA1-tBRCT pathogenic mutations S1655F (SF) and M1775R (MR) (Fig. 3 ), which reportedly destabilize the BRCA1-tBRCT domain 34–37 , impair SETX-BRCA1 interaction. Even though the pathogenicity of BRCA1-SF and MR variants has been attributed to defective homology-directed DNA repair 46–48 , our findings, together with a recent study demonstrating a role of the BRCA1-tBRCT domain in R-loop avoidance at rDNA loci 49 , implicate impaired R-loop resolution as a contributing factor to the pathogenicity incurred by the salient tBRCT mutations. We have leveraged the purified protein factors and in vitro systems that we have developed to delineate the mechanistic underpinnings of the SETX-BRCA1-BARD1 axis in R-loop resolution. Firstly, our biochemical studies have shown that BRCA1-BARD1 binds R-loops with nanomolar affinity (Fig. 4 a) and stimulates R-loop unwinding by SETX (Fig. 4 b). Importantly, while both BRCA1-BARD1 and BRCA1 ΔBRCT -BARD1 bound R-loops with comparable affinities, the latter is not only defective in SETX stimulation, but is in fact inhibitory in this regard. Furthermore, we have found that RAD52, which also interacts with SETX (Extended Data Fig. 3 e) and binds R-loops with affinities comparable to BRCA1-BARD1 (Extended Data Fig. 3 d), inhibits R-loop dissociation by SETX (Fig. 4 c), further demonstrating specificity of the stimulatory effect of BRCA1-BARD1 on SETX. Moreover, we have shown that BRCA1-BARD1 alleviates the inhibitory effect of RAD52 on SETX R-loop unwinding (Fig. 4 d). Based on these new findings, we propose a model (Extended Data Fig. 7 ) where BRCA1-BARD1 (i) forms a physical complex with SETX and promotes SETX-mediated R-loop unwinding and (ii) helps overcome the inhibition imposed by RAD52. Based on this study and previous reports of RAD52 functioning in transcription-associated homologous recombination 23,24 , we speculate that RAD52-dependent repair comes into play when R-loops are converted into DSBs. In this regard, BRCA1-BARD1-dependent engagement and dissociation of R-loops help prevent RAD52 accumulation and DSB generation (Extended Data Fig. 7 ). In support of this hypothesis, we have shown that the SETX-SA phospho-null mutant that is impaired for BRCA1 interaction fails to be recruited to R-loops, and as a result, excessive R-loops (Fig. 5 a,b), RAD52 foci, and DSBs accumulate in cells expressing this mutant (Fig. 5 c,e). Interestingly, even though the catalytic null SETX-EA mutant remains robustly recruited to R-loops, cells expressing this mutant also exhibit R-loop accumulation as well as elevated RAD52 foci and DSBs. We note that cells depleted of SETX or expressing the EA or SA are also sensitized to PARPi (Fig. 5 f). Interestingly, PARP1 is recruited to R-loops, and inhibition of PARP activity or its depletion leads to increased R-loop accumulation and genomic instability 50 . This could explain why PARP1 inhibition in cells lacking SETX activity induces synthetic lethality as we have shown here. As such, tumors harboring mutations that abrogate SETX activity or confer defects in SETX-BRCA1 interaction may be targeted via PARPi. R-loops reportedly block replication machinery via head-on conflicts 42 , and lead to gaps in the nascent DNA strands via mechanisms suggested in other studies 51,52 . Even though yeast Sen1 protein has been reported to localize to replication forks to promote fork progression and maintain genome stability 21,53 , an analogous role of SETX has not been demonstrated in human cells. Importantly, our iPOND and SIRF-PLA analyses revealed the presence of SETX at replication forks in an R-loop dependent manner (Fig. 6 a,c). The requirement of BRCA1 and SETX-phospho-Ser642 for replication fork association of SETX provides formal evidence for the SETX-BRCA1-BARD1 ensemble in the prevention of transcription-replication conflicts via R-loop resolution. Accordingly, SETX knockdown cells experience elevated transcription-replication conflicts, a phenotype that can be complemented by ectopic expression of SETX-WT, but not the SETX-SA or SETX-EA mutant (Fig. 6 c). Concordantly, mutant SETX cells experience elevated impediment of replication fork progression and increased fork stalling (Fig. 7 ) In summary, we have developed reconstituted systems to demonstrate that full length SETX has robust R-loop unwinding activity, physically interacts with BRCA1, and that BRCA1-BARD1 upregulates the R-loop unwinding activity of SETX. Altogether, these findings provide the molecular basis for elucidating the role of the SETX-BRCA1 axis in R-loop resolution and the promotion of replication fork procession in a transcribing genome, preventing generation of R-loop associated DSBs and replication stress. Declarations Acknowledgements We are grateful to Dr. Claus Azzalin at iMM, Universidade de Lisboa, for providing the telo-R-loop template plasmid, Dr. Lee Zou at Duke University for RAD52 K144A bacterial expression plasmid, and Dr. Bing Xia at Rutgers University for BRCA1-WT, ∆BRCT, S1665F and M1775R expression plasmids. We thank Dr. Hardeep Kaur (P.S. lab) for sharing important reagents. AD was supported by a Brown-Coxe postdoctoral fellowship from Yale University, a Jean B Kempner postdoctoral fellowship from University of Texas Medical Branch at Galveston, TX, and Office of Postdoctoral Affairs Fellowship from UT Health San Antonio, TX. This study was supported by National Institutes of Health grants RO1 ES007061, R35 CA241801, PO1 CA092584 (P.S.), RO1 CA168635 (G.M.K. and P.S.), R01GM141091 and R01CA268641 (W.Z.), U19AI150574, R01CA219836 (D.Z.), R50 CA265315 (Y.K.), P30 CA054174 (P.S., S.K.O. and D.Z.), R01 CA246807 (S.B.), R01 CA205224 (R.H.), R01 GM136717, R01 CA237286 (A.V.M.), R01CA152063 1R01CA241554 (A.J.R.B.), and PO1 CA092584 (P.S., A.V.C., D.T.M. and M.S.T.), by awards from the Cancer Prevention and Research Institute of Texas (CPRIT) REI RR210023 (A.V.M.), RP150445 (A.J.R.B.), RP210102 (W.Z.), and RP220269 (R.H.), by an award from the Congressionally Directed Medical Research Programs BC191160 (A.V.M.), by a Hyundai Hope on Wheels Scholar Grant (G.M.K.), by an American Cancer Society Research Scholar grant RSG-22-721675-01-DMC (W.Z.), and by a SU2C-CRUK award RT6187 (A.J.R.B.). The MST assays were carried out in the Center for Innovative Drug Discovery supported by CPRIT Core Facility Award RP210208 (D.Z.). P.S. is the holder of the Robert A. Welch Distinguished Chair in Chemistry (AQ-0012) and A.V.M. is the holder of the Joe R. and Teresa Lozano Long Chair in Cancer Research. The phosphopeptide mapping analysis was performed in the George L. Wright Center for Biomedical Proteomics supported by the Hampton Roads Biomedical Research Consortium. OJS holds the Anthem Distinguished Professorship in Cancer Research. Author Contributions A.D. generated and purified full length human SETX WT, point mutants and truncated proteins, BRCA1-BARD1 WT and truncated proteins, and RAD52 K144A , designed and performed all biochemical assays, in vitro pulldowns, and co-IP experiments. Y.K. purified human RPA and RAD52. J.H.J. performed the cell-based experiment including generation of stable cell lines, PLA, co-IP, S9.6 slot blot, foci formation experiments and clonogenic survival assay. Q.F., E.D. and B.D.L.P.A. performed the DNA fiber experiments. S.Z., supervised by D.Z. performed the MST experiments. F.L. performed the iPOND experiments. J.O.N. and O.J.S. were responsible for design and implementation of the mass-spectrometry-based studies. D.T.M., A.V.C. and M.S.T. generated BacMam-SETX and obtained affinity purified SETX from human cells for mass-spectrometric studies. A.B.T. provided insights on structural information on SETX Alphafold structure. W.L. generated HeLa-shBRCA1 cell line. A.O.H. performed some cell-based experiments. A.D., J.H.J., Y.K., G.K. and P.S. reviewed and participated in data interpretation together with other authors. A.D., W.Z., G.K. and P.S. conceived the project and developed experimental strategies. G.K., W.Z., and P.S. supervised and provided funding support. A.D. and P.S. wrote the manuscript with input from all authors. Declaration of Interests The authors declare no competing interests. Methods Protein Purification To express 6XHis-MBP-SETX-Flag, a 800 ml culture of Trichoplusia ni (Tni) insect cells (1x10 6 cells/ml, Expression Systems) in ESF921 media (Expression Systems) was infected with 8 ml baculoviral suspension followed by a 70 h incubation. Cells were collected by centrifugation and stored at -80°C until use. All the protein purification steps were carried out at 0-4°C. Cell pellet was resuspended in buffer L1 (50 ml per gram of pellet), composed of 50 mM Tris-HCl, pH 7.5, 500 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.01% NP-40, 2 mM ATP, 4 mM MgCl 2 , cOmplete protease inhibitor cocktail (MilliporeSigma), and 1 mM PMSF, with sonication. The lysate was clarified by ultracentrifugation at 100,000 x g for 45 min before being incubated with 1 ml Anti-FLAG-M2 affinity resin (MilliporeSigma) for 1 h. The resin was washed with 200 ml buffer W (50 mM Tris-HCl, pH 7.5, 500 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.01% NP-40, 2 mM ATP, 4 mM MgCl 2 ) and then with 50 ml buffer T1 (50 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.01% NP-40) with 100 mM KCl. SETX was eluted with 10 ml buffer T1 with 100 mM KCl, 0.25 mg/ml Flag peptide (Sigma) and cOmplete protease inhibitor cocktail (MilliporeSigma). The elution was fractionated in a HiTrap Q HP (1 ml) column (Cytiva) with a 30 ml gradient of 100-500 mM KCl in buffer T1. Peak fractions were concentrated using Amicon® Ultra Centrifugal Filters (100 kDa MWCO), divided into 2 µl aliquots, flash frozen in liquid nitrogen and stored at -80ºC. SETX mutants and truncated proteins were purified using the same procedure. A BRCA1(Flag)-BARD1(6XHis) MacroBac plasmid was generated via ligation independent cloning in 438a vector and recombinant baculovirus was generated in Sf9 insect cells. BRCA1-BARD1 was expressed in 800 ml Tni cells (1x10 6 cells/ml) and purified following our established method 33 . BRCA1 D BRCT -BARD1 was expressed and purified following the procedures developed for its wild type counterpart. GST-tagged BRCA1-tBRCT (residue 1528-1863) and BARD1-tBRCT (residue 423-777) were expressed in E coli BL21 Rosetta cells transformed with pGEX6p1-GST-BRCA1-tBRCT or pGEX6p1-GST-BARD1-tBRCT. Cells were grown in 6L cultures until OD 0.7 then protein expression was induced by the addition of 0.2 mM IPTG followed by a 18 h incubation at 16°C. All the subsequent steps were carried out at 0-4 o C. Cells were harvested by centrifugation, resuspended in 50 ml buffer T2 (50 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.1% NP-40) with 500 mM KCl, 2 mM PMSF, and cOmplete protease inhibitor cocktail), and lysed by sonicated. Clarified cell lysate was incubated with 1 ml Glutathione-Sepharose resin (Cytiva) for 1 h at 4°C, followed by washing with 200 ml buffer T2 with 50 mM KCl, 2 mM ATP, and 4 mM MgCl 2 . Then protein was released from the resin by cleaving the GST tag in an overnight incubation with 5 ml HRV-3C protease (Pierce) at 4°C. For obtaining GST-tagged proteins, elution was performed using buffer T2 with 500 mM KCl,10 mM glutathione, and cOmplete protease inhibitor. Proteins were further purified in HiTrapSP-HP with a gradient of 100-500 mM KCl in buffer T2, followed by size exclusion chromatography in a Superdex 200 column (Cytiva) in buffer T2 with 200 mM KCl. Peak fractions were concentrated, divided into 5 µl aliquots, flash frozen and stored at -80ºC. RAD52 (WT), RAD52 K144A 56 , and S. cerevisiae Rad52 were purified following our published procedures 57-59 . Generation of R-loop and other nucleic acid substrates For the construction of nucleic acid substrates, PAGE purified oligonucleotides (IDT) (Supplementary Tables 1 and 2), were labeled at their 5'-terminus with T4-Polynucleotide kinase (NEB) using γ- 32 P-ATP. Labelled oligonucleotides were annealed with an equimolar amount of unlabeled oligonucleotides (Supplementary Table 2) by gradually decreasing the reaction temperature from 95ºC to 4ºC in a thermal cycler. ATP hydrolysis assay A 50 µl stock ATP solution that contained 1 µl γ- 32 P ATP (10 µCi/µl, Perkin Elmer) and 100 µM of cold ATP was prepared. SETX-WT or mutant was incubated in 10 µl (final volume) buffer R1 (10 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 50 mM NaCl, 1 mM DTT, 1% glycerol) with 1 µl of the γ- 32 P ATP mix without or with 1 µM ssDNA (D7) or ssRNA (R1) at 37°C. The reaction was halted by mixing 2 µl of the reaction mixture and 2 µl of buffer S (1% SDS and 20 mM EDTA), and the level of ATP hydrolysis was determined by phosphorimaging analysis after thin layer chromatography in PEI-cellulose sheet (Sigma–Aldrich, Cat No. Z740237–25EA; 60 . R-loop binding assay The indicated protein species was incubated with 32 P-labelled R-loop in 10 µl buffer R2 (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1% glycerol, and 100 µg/µl BSA) for 30 min on ice. After adding 2 µl buffer LD (100 mM Tris–HCl, pH 7.5, 10 mM EDTA, 50% Glycerol, 0.15% Orange G), EMSA was carried out on 5% polyacrylamide gels in TAE buffer (30 mM Tris-acetate, pH 7.4 and 0.5 mM EDTA). The gels were dried onto Hybond-N membrane on top of Whatman filter paper and then subject to phosphorimaging analysis. Nucleic acid unwinding assay SETX-WT or mutant was incubated with 2 nM of the indicated 32 P-labelled substrate in 10 µl buffer R3 (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 2.5 mM MgCl 2 , 1.5 mM ATP, 10% glycerol, 0.2 µg/µl BSA, and 1 mM DTT), and 1 µl Rnasein (Promega; for RNA-DNA or R-loops) for 20 min at 37°C. The reactions were stopped by adding 1 µl 0.5% SDS and 1 µl proteinase-K (5 mg/ml) and a 5-min incubation at 37°C. After adding 2 µl buffer LD, reaction mixtures were resolved in 10% TAE native gels at 10°C. Gels were dried onto Hybond-N membrane on top of Whatman filter paper and then subject to phosphorimaging analysis. Generation of plasmid telo-R-loop and unwinding assay Telo R-loop was generated via in vitro transcription following a protocol modified from the literature 29 . Briefly, 2 µg pcDNA6-Telo plasmid containing a ~ 1 kb human telomeric repeat sequence was incubated with 2 µl T7 RNA pol (NEB) and ATP, CTP, GTP (2.5 mM each), 825 nM α-P 32 -UTP (3000 Ci/mmol; PerkinElmer), and 2 µl RNasein (Promega) in 1X T7 RNA pol buffer (NEB) for 30 min at 37°C. The reaction was stopped by adding cordycepin (5 mM) and, after a 10-min incubation, MgCl 2 (5 mM) was added to stabilize the R-loops. Telo-R-loop was purified with a Micro Spin S-400 column (Cytvia), aliquoted, and stored at -20°C. Telo-R-loop unwinding assay was conducted with 1 ng of the substrate and the indicated amount of SETX-WT or mutant in 10 µl of buffer R3 (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl 2 , 2 mM ATP, 10% glycerol, 0.2 µg/µl BSA, 1 mM DTT, and 1 µl Rnasein) at 37°C for 20 min. The reactions were stopped by adding 1 µl each of 0.5% SDS and proteinase-K (5 mg/ml) followed by a 5-min incubation at 37°C. Reaction mixtures were resolved on 0.9% agarose gels in TAE buffer. Gels were dried on Hybond-N membrane on top of Whatman filter paper and then subject to phosphorimaging analysis. Affinity pulldown assays For MBP pulldown experiments, 1 µM MBP-tagged SETX or SETX ΔHD was incubated with 1.5 µM BRCA1-BARD1 or BRCA ΔBRCT -BARD1 at 4°C for 30 min in 20 µl buffer M (25 mM Tris-HCl pH 7.5, 10% Glycerol, 0.5 mM EDTA, 0.05% Igepal CA-630, 1 mM DTT, and 150 mM KCl), with protein complexes being captured with 20 µl amylose resin (NEB). For GST pulldown experiments, 5 µM GST-tagged BRCA1-BRCT or BARD1-BRCT was incubated with 3 µM SETX ΔHD or SETX-HD at 4°C for 30 min in 20 µl buffer M, with protein complexes being captured with 20 µl glutathione sepharose 4 fast flow resin (Cytvia). The resin was washed three times with 100 µl buffer W containing 200 mM KCl before eluting bound proteins with 1X Laemmli buffer and a 4-min incubation at 65°C. The supernatant and eluate were analyzed by SDS-PAGE and Coomassie staining or western blotting. To test interaction of FAM-labelled SETX phospho-peptide and FLAG-tagged BRCA1-BARD1, or GST-tagged BRCA1-BRCT and BARD1-BRCT, 0.5 µM peptide was mixed with 1 µM of the indicated protein in buffer R1 for 30 min at 4 o C. Then, protein complexes were captured using either anti-FLAG-M2 resin or glutathione sepharose 4 fast flow resin by constant mixing for 30 min. After washing the resin three times with 100 µl buffer M containing 200 mM KCl, bound proteins were eluted 20 µl Laemmli buffer at 65°C for 4 min. The supernatant and eluate, 1 µl each, were spotted onto nitrocellulose membrane and imaged in Bio-Rad fluorescence imager. The same samples were also run on 7.5% protein gel and subjected to Western blot analysis. Microscale thermophoresis Purified BRCA1-tBRCT was serially diluted 1:1 with HEPES buffer saline (pH 7.5) up to 12 times in a 384-well plate, starting from the highest concentration available (final concentration: 6.8 µM – 3.3 nM). Following the addition of FAM-labeled SETX peptides (final concentration 10 nM for SPTF, APTF and SPTA; 20 nM for pSPTF, EPTF and DPTF), samples were loaded into regular glass capillaries (MO-AK002, Nanotemper Technologies GmbH, München, Germany). Microscale thermophoresis was then measured using a Nanotemper NT. Automated (Nanotemper Technologies GmbH, München, Germany). Fluorescent power was determined using the autodetection initially and set to 67%, 100%, 99%, 100%, 58%, 63% for SPTF, pSPTF, APTF, SPTA, EPTF and DPTF respectively, and IR laser power was set to medium level to apply the heat gradient to each capillary and light emitting diode power to track fluorescence migration along the heat gradient. Relative fluorescent data between 1.5s to 2.5s time window after IR laser onset was used for binding affinity evaluation. Data from 3 independent experiments (9 measurements for pSPTF) were fitted using the built-in models in the MO Affinity Analysis V2.3 software. The final data were plotted using GraphPad Prism 9.5. Mass spectrometric analysis of SETX Recombinant SETX was expressed in human Expi293F cells by infection with a BacMam baculovirus prepared from the pEZT-BM vector (Addgene #74099). The BacMam baculovirus was generated using the standard Bac-to-Bac procedures following manufacturer’s instructions (Thermo Fisher). For inducing phosphorylation of SETX, okadaic acid (Calbiochem) at 25 nM (final concentration) was added to cells at 44 h post infection and 1 µM CPT (Sigma) was added 1 h before cell harvesting. Cells were lysed in buffer L2 (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.3% NP-40, 10% glycerol) supplemented with 2 mM PMSF, 2 mM beta-mercaptoethanol, and protease inhibitors (ETDA-free protease inhibitor mini tablet (Millipore Roche), and Phosphatase inhibitor mini tablet (Thermo Fisher-Pierce)), with Dounce homogenization, followed by sonication. SETX-FLAG affinity purification was performed by incubating the clarified cell lysate with anti-Flag M2 resin (Sigma) at 4°C with gentle rocking for 1.5 h, followed by protein elution with 3x Flag peptide (APExBio) in buffer L2. Affinity purified SETX from human cells were precipitated with acetone, and separated by SDS-PAGE followed by Coomassie Blue staining. SETX containing gel slices were excised, cut into small 1mm 2 pieces, washed in solution A (50 mM NH 4 HCO 3 pH 8.0), destained in solution B (50 mM NH 4 HCO 3 /50% acetonitrile), dehydrated in solution C (100% acetonitrile), and dried in a vacuum concentrator. Proteins were reduced with 10 mM DTT in 50 mM NH 4 HCO 3 for 1 h at 56°C and alkylated with 55 mM iodoacetamide in 50 mM NH 4 HCO 3 for 1 h at room temperature in the dark. Then, pulverized gel pieces were washed (with solutions A, B, and C), dried in a vacuum concentrator, and rehydrated with trypsin (Promega V511A, 1:20 enzyme:substrate ratio) in solution D (50 mM NH 4 HCO 3 /10% acetonitrile) with a 30-min incubation on ice. Samples were covered with additional solution D and incubated overnight at 37°C. Peptides were extracted with 50% acetonitrile/0.1% formic acid. The digestion solution and peptide extracts were combined and dried in a vacuum concentrator. The pellet was resuspended in 0.1% formic acid and the peptide concentration determined with a NanoDrop One spectrophotometer (Thermo Fisher Scientific, 205 nm, Scopes method). The peptide concentration was normalized before LC-MS/MS. MS spectra were searched using MaxQuant software (version 2.1.0) against a human Swiss-Prot database (20,291 entries, canonical database). The following variable modifications were used for the searches: oxidation of methionine, deamidation of asparagine and glutamine, acetylation of protein N-termini and phosphorylation of serine, threonine and tyrosine residues. Carbamidomethylation of cysteine was included as a fixed modification. Trypsin was included as the protease with a maximum of two missed cleavage sites. The mass accuracy tolerances were set at 20 ppm for the first search and 10 ppm for the main search with a minimum peptide length of 7 amino acids. Match between runs was enabled with a match window of 0.7 min and alignment time of 20 min. The label-free quantification (LFQ) algorithm was used to calculate of LFQ intensities, and the LFQ minimum ratio count was set to 2. False discovery rates (FDR) for peptide spectral matches (PSM) and protein identifications were set at 1% (0.01). Complementary database searches for peptide and protein identification were performed using Mascot (Matrix Science). The relative abundance of non-phosphorylated and phosphorylated S642 in Non-CPT treated and CPT treated cells was determined using the LFQ intensity values obtained from MaxQuant. In a first step, the sum of normalized intensities values of all identified peptides (modified and unmodified) that contain S642 (DMHCLEASS*PTFSK, DMHCLEASS*PTFSKEPMK, DMHCLEASS*PTFSKEPMKVQDSVLIK, KDMHCLEASS*PTFSK and KDMHCLEASS*PTFSKEPMK) was obtained. The relative abundance of S642 phosphorylation in the two samples was calculated by determining the sum and proportion of the intensities of the peptides containing phosphorylated S642 compared to the summed intensity of the modified and unmodified peptides. Cell lines and culture The human HAP1 SETX wild type and knock-out (KO) cells were purchase from Horizon Discovery (HZGHC001565c005) and maintained in IMDM with 10% (v/v) FBS (Gibco) at 37°C. The human HEK293, HEK 293A, and HeLa cell lines were cultured in DMEM with 10% FBS (Gibco) at 37°C. 293A cell lines with stable expression of GFP (N-terminal) and FLAG (C-terminal) fused SETX wild type and point mutants were generated in presence of puromycin (2 µg/ml) selection, then GFP-positive cells were sorted by flow cytometry (BD science). Co-immunoprecipitation and Western blot HEK293 or HEK 293A cells were lysed with NETN buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, 50 U/ml MNase, 50 U/ml Benzonase) plus protease and phosphatase inhibitor cocktails (GeneDepot). Co-immunoprecipitation (co-IP) was carried out by incubating cell lysates with M2 FLAG-agarose resin (Sigma) at 4°C for overnight, followed by washes (3X) with buffer W and elution with Laemmli buffer. Co-IP samples were subjected to SDS-PAGE with 4–20% SDS-polyacrylamide gradient gels and immunoblotted with the indicated antibodies.8 Proximity ligation assay (PLA) To analyze co-localization of SETX at R-loops and co-localization of PCNA and RNAPII at replication-transcription conflict sites, proximity ligation assay (PLA) was carried out as per manufacturer’s protocol (Duolink In Situ Starter kits; DUO92101; Sigma-Aldrich). Briefly, U2OS cells were cultured in glass bottom dishes (SKU#801002; NEST). Cells were transfected with GFP-SETX-FLAG WT/EA/SA or EV (Fig. 5a), and with an endogenous SETX-3’ UTR-targeting siRNA (Fig. 6c), for 48 hrs, or cells were treated with camptothecin (CPT; 10 µM) for 1 hr (Extended data Fig. 6g). For analyze co-localization of GFP/S9.6 or SETX/S9.6, cells were washed with PBS and pre-permeabilized with 0.25% Triton X-100/PBS for 10 min and fixed with 2% formaldehyde/PBS for 10 min. For treatment of RNases, cells were washed with PBS and pre-permeabilized in 0.25% Triton X-100/PBS with RNase H (10 U), RNase III (10 U), RNase T1 (10 U), or RNase A (10 µg) for 30 min at 37°C, before being fixed with 2% formaldehyde/PBS for 10 min. Otherwise, for analyze co-localization of PCNA and RNAPII, cells were washed with PBS and fixed with 2% formaldehyde/PBS for 10 min and cells were incubated with 0.25% Triton X-100/PBS for 30 min. Cells were washed with PBS twice, then kept in PLA Duolink blocking buffer (1% BSA in PBS) for 1 h at 37°C. Next, to detect colocalization of GFP-SETX-FLAG WT/EA/SA and R-loops, the cells were incubated with anti-GFP (ab290; abcam) and anti-S9.6 (Cat# MABE1095; Millipore) antibodies in Duolink antibody dilution buffer at 4°C overnight. In situ PLA probes (anti-mouse plus and anti-rabbit minus) were diluted 1:5 in Duolink antibody diluent and incubated to detect anti-GFP (rabbit) and anti-S9.6 (mouse) antibodies for 1 hr at 37°C. After washing with buffer A (10 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween 20) three times for 5 min each, dishes were incubated with Duolink ligase at 37°C for 30 min and then washed with buffer A twice for 5 min each. Amplification mix prepared as per manufacturer’s instructions was added into each dish and incubated at 37°C for 100 min in the dark. Lastly, dishes were washed with buffer B (200 mM Tris, pH 7.5, 100 mM NaCl) three times for 5 min each and 0.01x buffer B once for 5 min. Doulink In Situ mounting solution with DAPI (4’,6-diamidino-2-phenylindole) was used to mount glass bottom dishes in PLA for nuclear staining and applied to an Olympus FV3000 confocal microscope. Intensity of foci was determined using the ImageJ (1.53a version; NIH) software. RNA/DNA hybrid slot blot assay To examine R-loop levels in HAP1-WT and HAP1-SETX-KO cells or in 293A cells stably expressing GFP and FLAG-tagged SETX-WT/EA/SA, where endogenous SETX was depleted with SETX 3′ UTR targeting siRNA, cells were lyzed with DNAzol (Invitrogen), and genomic DNA (gDNA) was sheared with sonication (50% of power; 10/20 sec interval of ON/OFF, 3X).The concentration of gDNA sheared was measured by Nano-photometer (N60; IMPLEN). The indicated concentration of gDNA was analyzed by the slot blot assay using Minifold I dot blot apparatus (Schleicher & Schuell). gDNA was treated with RNase H (10 U; NEB, M0297S), RNase III (10 U), RNase T1 (10 U), or RNase A (10 µg) at 37°C for 1 h to remove RNA/DNA hybrids. The membrane was blocked for 1 h at room temperate in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) with 5% milk and incubated overnight with the S9.6 antibody (Millipore; MABE1095) at 1:1000 concentration. Double-stranded DNA (dsDNA) antibody (ab27156; abcam) was used as a loading control of gDNA. The signals were detected by Western blot and quantified using the ImageJ software. Immunofluorescence HeLa cells were transfected with transiently expressing GFP and FLAG-tagged SETX-WT/EA/SA and endogenous SETX-3’ UTR targeting siRNA for 48 hrs. Cells were incubated with DMSO or PARP inhibitor (PARPi) for 4 h at 37C. Cells were fixed with 2% (v/v) formaldehyde in PBS for 15 min at room temperature and washed once with PBS and then, permeabilized with 0.5% Triton X-100 in PBS for 30 min. For R-loop detection via anti-S9.6 antibody in HAP1 SETX WT and KO cells, cells were pre-permeabilized with 0.25% Triton X-100 in PBS for 10 min, and then cells were fixed with 2% (v/v) formaldehyde in PBS for 15 min. Next, cells were blocked with 10% FBS/PBS for 20 min at room temperature and then treated with indicated antibodies in 10% FBS/0.25% Triton X-100 in PBS overnight at 4 o C. Cells were washed twice with 10% FBS/PBS and incubated with secondary antibody for 1 h at room temperature. Cells were mounted in Vectashield medium with DAPI (Vector Labs). Images were captured in an Olympus FV3000 confocal microscope and processed with the ImageJ software or Olympus Cellsens software. Clonogenic survival assay 293A cells stably expressing SETX-WT/EA/KA were transfected with SETX 3′-UTR targeting siRNA. After 2 days, cells were seeded at a density of 500 cells in 6-cm dishes. HAP1-WT and SETX-KO cells were seeded similarly. After being treated with CPT, cells were allowed to recover for 10 days. Colonies were fixed and stained with 0.5% crystal violet (C3886; Sigma). Viability was expressed as mean number of colonies obtained from three independent experiments. iPOND Analysis HeLa control and shBRCA1 cells 40 were cultured in DMEM containing 10% FBS medium. For BRCA1 knockdown, the cells were treated with 1 µg/ml doxycycline (Clontech Cat. No. 631311) for 3 days. RNaseH1-GFP was expressed 24 h prior to treatment with 1 nM Pladienolide-B (PB). The iPOND assay was performed as described previously (Sirbu, et al., 2012). Briefly, 1x10 8 cells were labeled with 10 µM EdU for 15 min at 37°C and washed once with EdU-free media. Then cells were treated with 1 nM PB for 24 h to induce R-loops. Following cross-linking treatment with 1% formaldehyde/PBS for 25 min, cells were quenched with 0.125 M glycine and washed with PBS three times. Collected cells were resuspended in permeabilization buffer (0.25% Triton-X/PBS) and incubated at room temperature for 30 min, then washed once with cold 0.5% BSA/PBS and PBS. Cells were collected by centrifugation and resuspended in click reaction buffer (10 µM Biotin-azide, 10 mM Na ascorbate, 2 µM copper(II) sulfate) and incubated with rotation at room temperature for 2 h. Cells were washed once with cold 0.5% BSA/PBS and PBS and collected by centrifugation. Cells were resuspended in buffer L3 (1% SDS with 50 mM Tris–HCl, pH 8.0) and a cocktail of protease inhibitors comprised of aprotinin, chymostatin, leupeptin, and pepstatin A) and subject to sonication. After centrifugation (16,000 x g for 10 min at room temperature), supernatant was collected, diluted with cold PBS to contain 0.5% SDS and 25 mM Tris-HCl (pH 7.5) and mixed with an equal volume of streptavidin-agarose resin for 20 h at 4 o C. The resin was washed once with 1 ml of cold lysis buffer, and then once with 1 M NaCl and twice with cold lysis buffer. Proteins were eluted with an equal volume of 2X Laemmli buffer at 95°C for 25 min. Western blotting was performed to analyze the eluate with anti-BRCA1(07-434, Millipore), anti-SETX (NB100-57542, Novus), anti-Histone H3 (C-2, sc-374669, Santa Cruz), anti-H2A.X (938CT5.1.1, sc-517336, Santa Cruz), and anti-PCNA (PC10, Novus) antibodies. In situ analysis of protein interaction at DNA replication forks (SIRF) HEK 293 cells stably expressing of GFP-SETX-FLAG WT/EA/SA in log phase were plated on glass bottom dishes and pulsed with 125 µM EdU in DMEM for 8 min and fixed with 2% paraformaldehyde (PFA) in PBS (pH 7.4) for 15 min at room temperature. Where HU treatment is indicated, EdU containing media was removed, dishes were washed twice with PBS, and incubated with DMEM containing 4 mM HU for 4 h before fixation. Next, cells were permeabilized with 0.25% Triton X-100 in PBS for 10 min at room temperature. Dishes were washed twice with PBS for 5 min. Cells were incubated in click-reaction cocktail (2 mM copper sulfate, 10 µM biotin-azide and 100 mM sodium ascorbate in PBS) for 1 h at room temperature. To observe localization of SETX-GFP or PCNA at replication forks, GFP-biotin and PCNA-biotin PLA was performed (see PLA section in the Star Methods), with primary anti-GFP or anti-PCNA and anti-biotin antibodies, respectively. Anti-Cyclin A2 (ab137769; abcam) antibody was used for detection of S-phase positive cells. DNA Fiber assay DNA fiber assay was performed as described 61 . Briefly, 293T cells stably expressing GFP and FLAG-tagged SETX-WT/EA/SA were sequentially pulse labelled with the nucleotide analogs CIdU (100 µM, Sigma-Aldrich, C6891) and IdU (100 µM, Sigma-Aldrich, I7125), according to the schematic in each Fig.. Where indicated, 48 h prior to pulse labelling, cells were transfected with pEGFPN1-RNAseH using the Fugene HD transfection reagent (Promega) and with control siRNA or that targeting SETX using Lipofectamine RNAiMAX (Invitrogen). After labelling, cells were collected and resuspended in Trypsin-EDTA (Sigma-Aldrich, T3924) at 3.5x10 5 cells per agarose plug. The preparation of agarose plugs, proteinase K treatment of plug, plug washing, and β-agarase treatment of melted plug and fiber combing were performed as described 61 . Immunodetection was carried out using rat anti-BrdU (BU1/75 (ICR1) antibody (Abcam, ab6326)) at a dilution of 1:20 for CldU detection, and mouse anti-BrdU (Clone B44) (BD Bioscience, 347580) at a dilution of 1:5 for IdU detection. Goat anti-rat 488 (Invitrogen, A11006) and donkey anti-mouse Cy3 (Invitrogen, A31570) were used as secondary antibodies at a dilution of 1:100. To assess replication fork restart, 1.2x10 6 cells were used per agarose plug. Cells were initially labeled with CIdU for 30 min, followed by treatment with 1 µM CPT for 30 min, and then labeled with IdU for an additional 30 min. Fibers were imaged in an Olympus Fluoview FV3000 Confocal Laser Scanning Microscope and quantification was with the ImageJ software. 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1","display":"","copyAsset":false,"role":"figure","size":812147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePurification and characterization of R-loop binding and unwinding activities of human SETX. (a) \u003c/strong\u003eFunctional domains in SETX: N-terminal domain (NTD; green), helicase domain (blue), with conserved motifs I, Ia, II, III, V and VI; walker-A motif mutant, K1969A (KA), walker-B motif mutant, E2182A (EA), breast cancer associated somatic mutations, N2010I (NI), and H2197R (HR). Putative disordered regions are highlighted in red. \u003cstrong\u003e(b)\u003c/strong\u003e SDS PAGE of purified full length human SETX species. SETX-wild type (WT), K1969A: (KA), E2182A (EA), H2197R (HR), and N2010I (NI). M: molecular weight markers. \u003cstrong\u003e(c)\u003c/strong\u003e Analysis of ATP hydrolysis by SETX (20 nM) with the indicated nucleic acid (1 mM 60 nt ssRNA or ssDNA). \u003cstrong\u003e(d\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eATP hydrolysis activity of SETX-WT, KA, EA, NI and HR (50 nM) tested with 1 mM RNA. \u003cstrong\u003e(e) \u003c/strong\u003eR-loop unwinding activity of SETX-WT (lanes 2-5; 6.25,12.5, 25 nM) and the mutants, EA, KA, NI and HR (lanes 6 to 9; 25 nM) tested with R-loops with a 5’-RNA overhang (left panel), or a 3’-RNA overhang (right panel). \u003cstrong\u003e(f) \u003c/strong\u003eSchematic of plasmid-based telo-R-loop created via \u003cem\u003ein vitro\u003c/em\u003e transcription and R-loop unwinding assay (top). Analysis of telo R-loop (50 pM) unwinding activity of SETX (lanes 2-6: 3, 6.25, 12.5, 25, 50 nM; lanes 7-8: 50 nM). Data from three independent experiments were quantified; % R-loop unwinding was calculated after subtracting the lane #1 band intensity as background signal from all lanes in each panel (e-f), and mean values ± SD were plotted; statistical evaluation was performed via Welch’s two-tailed t-test; P-values 0.0003-0.0007 and \u0026lt;0.0001 are represented as “ *** ”, and “ **** ”; HD; Heat-denatured substrate.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833044/v1/384135d6f1e307ab99a62162.jpg"},{"id":49602556,"identity":"a7c77085-46da-4d4d-b009-c86073e3d97f","added_by":"auto","created_at":"2024-01-15 05:45:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":672267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDefining the interaction between SETX and BRCA1.\u003c/strong\u003e \u003cstrong\u003e(a) \u003c/strong\u003eCo-IP analysis to test SETX interaction with BRCA1-BARD1 in lysates of human 293T cells. Cells were treated with 1 µM CPT for 1 h, as indicated. \u003cstrong\u003e(b) \u003c/strong\u003eMBP-tag pulldown to test interaction between purified SETX and BRCA1-BARD1. The supernatant (Sup) and SDS eluate fractions were analyzed by SDS-PAGE and Coomassie Blue staining. \u003cstrong\u003e(c, d) \u003c/strong\u003eMBP-tag pulldown to test SETX interaction with BRCA1-BARD1 (B1D1) or BRCA1\u003csup\u003e∆BRCT\u003c/sup\u003e-BARD1 (B1\u003csup\u003e∆B\u003c/sup\u003eD1) \u003cstrong\u003e(c)\u003c/strong\u003e, and SETX or SETX\u003csup\u003e∆HD\u003c/sup\u003e interaction with B1D1 or B1\u003csup\u003e∆B\u003c/sup\u003eD1 \u003cstrong\u003e(d)\u003c/strong\u003e. Supernatant (S) and eluate (E) fractions were analyzed by Western blotting with anti-MBP, anti-FLAG and anti-6XHis antibodies. \u003cstrong\u003e(e, f) \u003c/strong\u003eGST-tag pulldown to test SETX\u003csup\u003e∆HD\u003c/sup\u003e or SETX-HD interaction with GST-tagged BRCA1-tBRCT or BARD1-tBRCT. The supernatant (S) and eluate (E) fractions were analyzed by Western blotting with anti-MBP and anti-GST antibodies \u003cstrong\u003e(e\u003c/strong\u003e) or Coomassie Blue staining (\u003cstrong\u003ef\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833044/v1/eedbb0988dd39ba9ecfb2a2a.jpg"},{"id":49602330,"identity":"5203bccb-f85d-4f8a-82d8-0dfe4f79efc6","added_by":"auto","created_at":"2024-01-15 05:37:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1009345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhosphorylation of SETX Ser642 and its importance in SETX-BRCA1 interaction. (a) \u003c/strong\u003eSchematic of SETX interaction with BRCA1-BARD1. HD: Helicase Domain, R: Ring domain, DBD: DNA Binding Domain, CC: Coiled Coil, BB: tBRCT domain. Key residues for SETX-BRCA1 interaction are highlighted. \u003cstrong\u003e(b)\u003c/strong\u003e Sequence alignment showing conservation of SETX amino acid residues including Ser642 and Phe645 of the conserved SPTF motif. \u003cstrong\u003e(c)\u003c/strong\u003e MST analysis of BRCA1 and SETX interaction using BRCA1-tBRCT and FAM-labelled SETX SPTF peptide and those with S642 is replaced with phospho-S, or phosphomimic residues E or D, namely pSPTF, EPTF and DPTF, respectively; SPTF did not show BRCA1-tBRCT binding\u003cstrong\u003e; \u003c/strong\u003eK\u003csub\u003ed \u003c/sub\u003e[95%CI], pSPTF = 206 nM; CI indicates confidence interval. \u003cstrong\u003e\u0026nbsp;(d) \u003c/strong\u003eFLAG-tag co-IP analysis of interaction of FLAG-SETX-WT or S642A (SA) with Myc-tagged BRCA1 expressed in HEK293 cells, with or without 1 mM CPT treatment. \u003cstrong\u003e(e)\u003c/strong\u003e FLAG-tag co-IP analysis of FLAG-SETX interaction with Myc-tagged BRCA1-WT, S1655F, M1755R and DBRCT, transiently expressed in HEK293 cells. EV: Empty Vector.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833044/v1/60f44c5fb3823e4956122d8c.jpg"},{"id":49602332,"identity":"39435a8c-38b2-443f-ab47-44dc339e7643","added_by":"auto","created_at":"2024-01-15 05:37:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":796227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eR-loop binding of BRCA1-BARD1 and its effect on SETX R-loop unwinding. (a) \u003c/strong\u003eEMSA analysis of R-loop (2 nM) binding by BRCA1-BARD1 or BRCA1\u003csup\u003eDBRCT\u003c/sup\u003e-BARD1 (6.25, 12.5, 25, 50 nM). Unwinding of telo-R-loop (50 pM) by \u003cstrong\u003e(b)\u003c/strong\u003e SETX (10 nM) in the presence of BRCA1-BARD1 or BRCA1\u003csup\u003eDBRCT\u003c/sup\u003e-BARD1 (25, 50 nM), \u003cstrong\u003e(c)\u003c/strong\u003e SETX (50 nM) in presence of RAD52\u003csup\u003eWT\u003c/sup\u003e or RAD52\u003csup\u003eK144A\u003c/sup\u003e (2.5, 5, 10 nM), and \u003cstrong\u003e(d)\u003c/strong\u003e SETX (5 nM) activity in the presence of BRCA1-BARD1 (25, 50 nM) and RAD52 (5 nM), as indicated. Data from three (a-c), or two (d) independent experiments were quantified and mean value ± SD plotted; statistical evaluation was performed via Welch’s two-tailed t-test; P-values \u0026lt; 0.05 and ≤ 0.005 are represented as “ * ” and “ ** ”, respectively ; n.s. indicates not significant.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833044/v1/c7bfeb27bd28491ab60b899f.jpg"},{"id":49602331,"identity":"ce7adfc1-5183-4fd0-bb44-619f5b432813","added_by":"auto","created_at":"2024-01-15 05:37:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1546558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of SETX-S642 phosphorylation in R-loop resolution and genome maintenance. (a) \u003c/strong\u003eRepresentative micrographs of\u003cstrong\u003e \u003c/strong\u003eGFP-S9.6 PLA results showing colocalization of GFP-tagged SETX-WT, EA or SA\u003csup\u003e \u003c/sup\u003eand R-loops via transient transfection of GFP-tagged SETX-WT, EA or SA constructs in U2OS cells. An empty vector expressing only GFP (EV) was used a negative control; number of PLA foci per cell are plotted in the right panel. Scale bars, 10 μm. Data represent mean ± SEM of two independent experiments (n≥100). \u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, and \u003cem\u003eP \u003c/em\u003e\u0026lt;0.001 versus the control are represented by “ * ” and “ ****\u0026nbsp; ”, respectively;\u0026nbsp; n.s., not significant. \u003cstrong\u003e(b)\u003c/strong\u003e S9.6 slot-blot analysis of R-loop accumulation in HEK293. EV, SETX-WT, EK or SA stable cells, transfected with control siRNA (siCTRL) or 3' UTR-targeting SETX siRNA (siSETX-3' UTR), as indicated; anti-dsDNA antibody was used as a loading control. S9.6/dsDNA ratio was plotted in the bottom panel. Data represent mean ± SEM of three independent experiments. \u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, and \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01 versus the control are represented by “ * ” and “ **\u0026nbsp; ”, respectively;\u0026nbsp; n.s., not significant. \u003cstrong\u003e(c)\u003c/strong\u003e Representative micrographs of\u003cstrong\u003e \u003c/strong\u003eγH2AX and RAD52 foci in overexpression of EV, SETX-WT, EK or SA in HeLa cells, transfected with siCTRL or siSETX-3' UTR, as indicated, after treatment with DMSO or Olaparib (10 μM, 4 h); the intensity of γH2AX and RAD52 in each cell was quantified and plotted in panels \u003cstrong\u003e(d) \u003c/strong\u003eand \u003cstrong\u003e(e), \u003c/strong\u003erespectively\u003cstrong\u003e. \u003c/strong\u003eScale bars, 10 μm. Data represent mean ± SEM of three independent experiments (n≥100). “ **** ”\u0026nbsp; denotes \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus the control; n.s., not significant.\u003cstrong\u003e (f)\u003c/strong\u003e Olaparib sensitivity of HEK293 EV, SETX-WT, EK or SA stable cells, transfected with siCTRL or siSETX-3' UTR, as indicated. Data represent mean ± SEM of three independent experiments. “ ** ” denotes \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the control; n.s., not significant.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833044/v1/f5dc28cff647c62e34c366e1.jpg"},{"id":49602702,"identity":"378300a5-041d-4c57-a4d7-cede74e5b534","added_by":"auto","created_at":"2024-01-15 05:53:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2138180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of Ser642 phosphorylation in SETX recruitment to replication forks and resolution of transcription-replication conflicts.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e iPOND analysis of SETX, BRCA1, gH2AX, PCNA and Histone H3 recruitment at replication fork in HeLa and shBRCA1-HeLa cells, with RNaseH1 overexpression, doxycycline (Dox)-induced BRCA1 depletion and pladienolide-B (PB; 1 nM, 24 h) treatment as indicated. No-click: without EdU biotin-azide click reaction. \u003cstrong\u003e(b)\u003c/strong\u003eSIRF analysis of SETX-WT, EA or SA colocalization at replication forks in HEK293 EV, SETX-WT, EK or SA stable cells; SIRF intensity in Cyclin A2 (CycA2)positive S-phase cells was plotted in the right. Scale bars, 10 μm. Data represent mean ± SEM of three independent experiments (n≥100). “ * ” and “ **** ” denote \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, respectively, versus the control; n.s., not significant. \u003cstrong\u003e(c)\u003c/strong\u003e Analysis of transcription-replication conflicts via PCNA-RNA pol II (RPII) PLA in cells treated with siCTRL or siSETX-3' UTR and those expressing GFP-tagged SETX-WT, EA or SA in U2OS cells, as indicated. +RH1, RNase H1-treated for 1 h before fixation. Scale bars, 10 μm. Data represent mean ± SEM of two independent experiments (n≥100). “ **** ” denotes \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus the control. \u003cstrong\u003e(d)\u003c/strong\u003e Analysis of replication fork progression via PCNA-EdU PLA in cells treated with siCTRL or siSETX-3' UTR in HEK293 EV, SETX-WT, EK or SA stable cells, as indicated. +HU, Hydroxyurea-treated cells. Anti-Biotin antibody was used to detect conjugates of Edu-azide-Biotin. Scale bars, 10 μm. Data represent mean ± SEM of three independent experiments (n≥100). “ **** ” denotes \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001versus the control.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833044/v1/9078b207d5eed33f4e1491fe.jpg"},{"id":49602557,"identity":"9e532cfc-7698-48f7-9e2e-fd2bd3495a37","added_by":"auto","created_at":"2024-01-15 05:45:45","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":695055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of SETX in replication fork progression\u003c/strong\u003e. \u003cstrong\u003e(a)\u003c/strong\u003e Schematic of replication fork progression assay with CldU (100 μM) and IdU (100 μM) labelling for 30 min each, and representative micrographs of DNA fiber are shown. \u003cstrong\u003e(b)\u003c/strong\u003e The ratio of IdU to CldU tract length plotted for the indicated conditions. Data represent mean ± SD of two independent experiments\u003c/p\u003e\n\u003cp\u003e(n=100-227). \u003cstrong\u003e(c)\u003c/strong\u003e The relative percentage of stalled forks. Data represent mean ± SD of two independent experiments (n=220-609). \u003cstrong\u003e(d)\u003c/strong\u003e Schematic of replication fork restart assay with 100 μM CldU labelling followed by CPT (1 μM) treatment and then IdU (100 μM) labelling, for 30 min each, and representative images of DNA fiber are displayed. \u003cstrong\u003e(e)\u003c/strong\u003e The ratio of IdU to CldU tract length plotted for the indicated conditions. Data represent mean ± SD of two independent experiments (n=70-167). Statistical analysis was performed using Fisher’s exact t-test. \u003cstrong\u003e(f)\u003c/strong\u003e The relative percentage of restart fibers. Data represent mean ± SD of two independent experiments (n=300-1408). Statistical analysis was conducted using Fisher's exact test. Significant differences are indicated with “ * ” (\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05), \u0026nbsp;“ *** ” (\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001), and “ **** ” (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833044/v1/11a71ecc88c0167722f8136b.jpg"},{"id":105886457,"identity":"6613b5f1-792f-4f84-bb6e-34edbfe23ba2","added_by":"auto","created_at":"2026-04-01 07:29:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9227828,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3833044/v1/04215c3d-211a-4f14-ae26-8564f0c420a4.pdf"},{"id":49602333,"identity":"3cf97147-78a5-49b2-99ce-c5714e23a401","added_by":"auto","created_at":"2024-01-15 05:37:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":143658,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Tables\u003c/p\u003e","description":"","filename":"SupplementalInformationNSMB.docx","url":"https://assets-eu.researchsquare.com/files/rs-3833044/v1/1dbebe4a61313f9c1b396b9c.docx"},{"id":49602336,"identity":"bd643519-3644-4f31-9d05-0cceebba7886","added_by":"auto","created_at":"2024-01-15 05:37:45","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3597718,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFigs.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3833044/v1/b2f31f1cb08db43f5b4521ee.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mechanism of SETX-BRCA1-BARD1 complex in resolution of R-loops and transcription-replication conflicts","fulltext":[{"header":"Introduction","content":"\u003cp\u003eR-loops are three-stranded nucleic acid structures that harbor a RNA transcript hybridized to a DNA template and serve important physiological roles, including gene expression regulation and Ig class-switch recombination\u003csup\u003e1,2\u003c/sup\u003e. However, excessive R-loops can compromise genome stability as they impede the progression of replication forks in dividing cells and can lead to the induction of DNA double strand breaks (DSBs) via nucleolytic cleavage of the ssDNA within the R-loop structure\u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. Senataxin (SETX), a putative RNA-DNA helicase that promotes transcription termination\u003csup\u003e5\u003c/sup\u003e, was identified as a BRCA1 interactor in a proteomic analysis\u003csup\u003e6\u003c/sup\u003e. Subsequent studies have implicated the SETX-BRCA1 complex in the maintenance of genomic stability at transcribing genomic regions via R-loop removal\u003csup\u003e7\u003c/sup\u003e. SETX accumulates at DSBs in a transcription-dependent manner, and provides resistance against oxidative stress and genotoxic agents such as camptothecin (CPT) and mitomycin C (MMC)\u003csup\u003e8,9\u003c/sup\u003e. SETX plays a role in the homology-directed repair of DSBs\u003csup\u003e10\u003c/sup\u003e. Depletion of SETX or BRCA1 also leads to increased accumulation of cytosolic RNA-DNA hybrids and activation of innate immune signaling via cGAS-STING pathway due to XPG-mediated processing of nuclear R-loop\u003csup\u003e11\u003c/sup\u003e. Hereditary mutations in SETX are associated with two motor neuron diseases \u0026ndash; a rare juvenile form of amyotrophic lateral sclerosis (ALS4)\u003csup\u003e12\u003c/sup\u003e, and ataxia oculomotor apraxia type 2 (AOA2)\u003csup\u003e13\u003c/sup\u003e, where pathogenicity has been largely associated with dysregulation of R-loop homeostasis in neurons\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSETX is a very large protein of 2,677 amino acid residues (303 kDa). It possesses within its C-terminal region (residues 1736\u0026ndash;2464) a domain conserved within the Superfamily 1B (SF1B) helicases. It also harbors a large region (residues 596\u0026ndash;1735) predicted to be disordered (Fig.\u0026nbsp;1a). The helicase core domain of SETX is structurally homologous to yeast Sen1\u003csup\u003e15\u003c/sup\u003e, which can unwind RNA-DNA a 5\u0026prime; to 3\u0026prime; direction with regards to the RNA strand\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e, to facilitate transcription termination\u003csup\u003e18\u0026ndash;20\u003c/sup\u003e, maintain genome stability via resolution of RNA-DNA hybrids at replication forks, and prevent transcription-replication conflicts\u003csup\u003e21,22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo elucidate the R-loop resolution mechanism of human SETX and its possible functional synergy with BRCA1, we have developed systems for expressing and purifying full length human SETX to near homogeneity (Fig.\u0026nbsp;1b) for biochemical testing. We demonstrate that SETX possesses a helicase activity that efficiently unwinds R-loops, and that the breast cancer associated mutations N2010I and H2197R impair SETX activity (Fig.\u0026nbsp;1d,e). Importantly, we also present data to show that SETX physically interacts with BRCA1 through the tandem BRCT domain of the latter and that SETX-BRCA1 complex formation is enhanced by the phosphorylation of S642 in SETX within the context of a SPTF motif. Our \u003cem\u003ein vitro\u003c/em\u003e studies demonstrate BRCA1 in complex with its obligatory partner BARD1 binds R-loops and stimulates SETX-catalyzed R-loop dissociation that is dependent on the interaction between SETX and BRCA1. On the contrary, SETX-mediated R-loop dissociation is impeded by RAD52, a homologous recombination factor that has been implicated in both BRCA1-dependent\u003csup\u003e23\u003c/sup\u003e and BRCA1-independent transcription associated DSB repair processes\u003csup\u003e24\u003c/sup\u003e. Importantly, this inhibitory effect of RAD52 is efficiently overcome by BRCA1-BARD1. We further demonstrate that human cells expressing SETX-catalytic mutants and the BRCA1 interaction defective S642A mutant accumulate R-loops and DSBs, and also exhibit hypersensitivity to Olaparib, an inhibitor of polyADP ribose polymerases (PARPs). Interestingly, increased RAD52 foci in cells expressing SETX mutants suggest that R-loop associated DSBs undergo repair via a RAD52-dependent mechanism in such a cellular setting. Finally, we show that SETX is recruited to stressed replication forks in a BRCA1-depdendent manner and is indispensable for replication fork progression. Altogether, our findings shed light on the molecular mechanism of the SETX-BRCA1-BARD1 axis in R-loop resolution and replication fork maintenance.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\n\u003ch2\u003eNucleic acid unwinding activities of SETX\u003c/h2\u003e\n\u003cp\u003eSETX harbors Walker A and Walker B motifs associated with ATP binding and hydrolysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea)\u003csup\u003e25\u003c/sup\u003e. To obtain human SETX, we constructed a recombinant baculovirus to express the full-length protein with N-terminal 6X-His-MBP and C-terminal FLAG tags in insect cells. SETX thus expressed is soluble and can be purified to near homogeneity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). Purified SETX hydrolyzes ATP with a strong dependence on either RNA or DNA, but shows no preference for either nucleic acid species with a K\u003csub\u003ecat\u003c/sub\u003e of ~\u0026thinsp;180 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). We asked whether SETX binds R-loops using the electrophoretic mobility shift assay (EMSA) with two different R-loop substrates having either a 5\u0026prime;-RNA overhang or no overhang. SETX bound these R-loops with a comparable affinity (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\n\u003cp\u003eWe generated two SETX mutants, SETX-K1969A (KA) and SETX-E2182A (EA), by mutating the highly conserved Lys1969 in the Walker A motif and Glu2182 in the Walker-B motif to Alanine (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea,b, Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb,c). As expected, both the SETX-KA and SETX-EA mutants failed to hydrolyze ATP (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). Next, we searched for pathological SETX mutations in the cBioPortal\u003csup\u003e26\u003c/sup\u003e, a cancer somatic mutation database, particularly focusing on the mutations occurring within the helicase domain, and chose two metastatic breast cancer-associated mutations N2010I (NI) and H2197R (HR) for mechanistic characterization. N2010 is located in motif Ia of SETX that is likely involved in nucleic acid binding, and His2197 is located between motif II/Walker-B and motif III likely required for ATP binding and hydrolysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb,c). Interestingly, the N2010S and H2197R SETX mutations are associated with the neurodegenerative disease ataxia with oculomotor apraxia type 2\u003csup\u003e27,28\u003c/sup\u003e, indicating that these genetic alterations have pathogenic consequences. SETX-NI and SETX-HR mutant proteins were purified from insect cells to near homogeneity following the same purification scheme as SETX-WT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). The results showed that both pathogenic mutations engender a defect in ATP hydrolysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eNext, we tested SETX activity on R-loops generated with oligonucleotides that harbor a 5\u0026prime; or 3\u0026prime; RNA overhang or without any such overhang (Supplementary Tables\u0026nbsp;1 and 2). SETX unwound all three R-loop substrates when ATP was present (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee, Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea) but not when ATP was substituted with the nonhydrolyzable ATP analog AMP-PNP (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed, lanes # 6, 12), indicating that R-loop unwinding is driven by ATP hydrolysis. SETX activity was the strongest on the R-loop substrate with a 5\u0026prime;-RNA overhang, followed by the substrate with a 3\u0026prime;-RNA overhang, then that with no overhang (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee, Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). Importantly, the Walker-domain mutants SETX-KA and SETX-EA and pathogenic mutants SETX-NI and SETX-HR all displayed defects in R-loop unwinding (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e\n\u003cp\u003eTo further define the substrate specificity of SETX helicase activity, we also tested duplex DNA, RNA-DNA, and R-loop substrates with a 30-nt 5\u0026prime; or 3\u0026prime; single stranded (ss) DNA or RNA overhangs (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea,b). Importantly, the results showed that SETX also unwinds D-loop (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, lane # 26\u0026ndash;30) and dsDNA with either a 5\u0026prime; or 3\u0026prime; overhang (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, lane # 1\u0026ndash;10), while it cannot unwind blunt-ended dsDNA (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Overall, the results indicated that SETX is more proficient at unwinding R-loops than any of the DNA-based substrates (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, SETX has only modest activity toward RNA-DNA substrates with either a 3\u0026prime; or 5\u0026prime; DNA or RNA overhang (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, lane # 11\u0026ndash;30), while it is devoid of the ability to unwind dsRNA with a 5\u0026prime; RNA overhang (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). We note that SETX is just as proficient in dissociating substrates that resemble R-loops but without the displaced ssDNA strand (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, lane # 16\u0026ndash;25). Altogether, our results indicate that SETX unwinds RNA-DNA hybrids embedded within dsDNA robustly and without any requirement for a displaced ssDNA strand.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eSETX activity on R-loops embedded within a plasmid\u003c/h2\u003e\n\u003cp\u003eHaving established that SETX is adept at dissociating synthetic R-loops constructed with oligonucleotides, we wished to determine whether SETX can also act on more physiologically relevant R-loop intermediates formed during transcription. For this, we generated \u003csup\u003e32\u003c/sup\u003eP -labeled R-loops via \u003cem\u003ein vitro\u003c/em\u003e transcription using T7 RNA polymerase. The template is embedded within a closed-circular dsDNA template containing\u0026thinsp;~\u0026thinsp;1 kb telomeric repeat sequence, referred to as pTelo (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef, schematics)\u003csup\u003e29\u003c/sup\u003e. Importantly, we found that SETX unwinds R-loops generated within the telomeric sequence robustly with a strict dependence on ATP hydrolysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef), but that all four SETX mutants, namely, K1969A, E2182A, N2010I and H2197R, are defective in this regard (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eDissection of SETX-BRCA1 interaction\u003c/h2\u003e\n\u003cp\u003eSETX was identified as a BRCA1-interacting factor in a proteomic analysis\u003csup\u003e6\u003c/sup\u003e. These two proteins co-immunoprecipitate from lysates of human cells and it has been suggested that BRCA1 and SETX function together in maintaining genome stability at transcription pause sites via R-loop resolution\u003csup\u003e7\u003c/sup\u003e. Other studies have also implicated BRCA1 in mitigating R-loop associated genome instability, which is linked with tumorigenicity\u003csup\u003e30,31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur own immunoprecipitation analysis using HEK293 cell lysate recapitulated co-precipitation of SETX with the BRCA1-BARD1 complex. The amount of coprecipitating SETX-BRCA1-BARD1 increased upon treatment of cells with CPT, a topoisomerase 1 (TOP1) inhibitor, that is known to enhance R-loop accumulation via generation of TOP1 cleavage complex and RNA Polymerase II (RNA Pol II) pausing (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea)\u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNext, we addressed whether SETX interacts with BRCA1-BARD1 directly. For this, we followed our published procedures to co-express BRCA1 and BARD1 in insects and purify the assembled complex to near homogeneity\u003csup\u003e33\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). Importantly, affinity pulldown analysis done in the presence of benzonase (a pan nuclease that digests both DNA and RNA) provided evidence that SETX directly interacts with BRCA1-BARD1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). Previously, SETX was reported to co-immunoprecipitate with a BRCA1 fragment (amino acid residues 1568\u0026ndash;1863) that encompasses the tandem BRCT (tBRCT) domain of BRCA1 \u003csup\u003e7\u003c/sup\u003e. To ask whether the BRCA1 tBRCT domain is required for SETX interaction, we generated BRCA1\u003csup\u003e\u0026Delta;BRCT\u003c/sup\u003e-BARD1 that lacks the BRCA1 tBRCT domain (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). Our pulldown result showed that BRCA1\u003csup\u003e\u0026Delta;BRCT\u003c/sup\u003e-BARD1 is indeed defective in SETX interaction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). To map the region in SETX that interacts with BRCA1-BRCT, we generated SETX-HD (amino acid residues 1932\u0026ndash;2677) and SETX\u003csup\u003e\u0026Delta;HD\u003c/sup\u003e (amino acid residues 1-1721) (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb) for pulldown analysis. we found that SETX\u003csup\u003e\u0026Delta;HD\u003c/sup\u003e interacts with BRCA1-BARD1 with an affinity comparable to full length SETX (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, lanes # 3\u0026ndash;6). Importantly, SETX\u003csup\u003e\u0026Delta;HD\u003c/sup\u003e failed to interact with BRCA1\u003csup\u003e\u0026Delta;BRCT\u003c/sup\u003e-BARD1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, lanes # 7\u0026ndash;10), but showed strong interaction with BRCA1-tBRCT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, lanes # 3\u0026ndash;4). However, SETX\u003csup\u003e\u0026Delta;HD\u003c/sup\u003e exhibited little or no affinity toward the BARD1 tBRCT domain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, lanes # 5\u0026ndash;6), which aptly underscores the specificity of the SETX-BRCA1\u003csup\u003etBRCT\u003c/sup\u003e interaction. Finally, we observed that SETX-HD also interacts with the BRCA1-tBRCT domain, albeit only weakly (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef, lane # 6). Altogether, our domain mapping effort provided evidence that SETX forms a complex with BRCA1-BARD1 via two interaction regions, one located within the N-terminal half of the protein, and other within its helicase domain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRole of SETX-Ser642 phosphorylation in SETX-BRCA1 interaction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA number of BRCA1 interacting factors, such as BACH1/FANCJ, Abraxas, and CtIP, relies on a consensus SPTF motif, in which the Ser residue is subject to phosphorylation, for complex formation with the tBRCT domain of BRCA1\u003csup\u003e34\u0026ndash;37\u003c/sup\u003e. Importantly, SETX possesses a S\u003csup\u003e642\u003c/sup\u003ePTF motif that is highly conserved in mammals but not in lower metazoans (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea,b). Phosphorylation of Ser642 in SETX peptides was reported in multiple whole cell phospho-proteomic studies\u003csup\u003e38,39\u003c/sup\u003e. Our own mass-spectrometric analysis of FLAG-affinity purified SETX expressed in Expi293F cells (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea-c), revealed that SETX-Ser642 is phosphorylated constitutively, with the extent of phosphorylation increased upon treatment of cells with CPT (Supplementary Tables\u0026nbsp;3, 4). Moreover, with a custom rabbit polyclonal antibody (Biomatik) against the SETX phospho-peptide CLEAS[\u003cstrong\u003epS\u003c/strong\u003e]PTFSKEPM, we showed that CPT treatment of insect cells led to an elevation of Ser642 phosphorylation level in recombinant SETX as well (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eNext, we examined the association of a carboxyfluorescein (FAM)-labelled SETX phospho-SPTF peptide with BRCA1-BARD1 and the tBRCT domain from either BRCA1 or BARD1 (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). Specifically, we incubated the labeled SETX peptide with either FLAG-tagged BRCA1-BARD1, or GST-tagged BRCA1-tBRCT and BARD1-tBRCT, followed by affinity pulldown via the affinity tags and analyzed eluates from the affinity matrices by dot blot or immunoblot analysis (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). We found that the SETX phospho-peptide interacts with full-length BRCA1-BARD1 and BRCA1-tBRCT, but not BARD1-tBRCT. We also employed microscale thermophoresis (MST) to independently verify conclusions drawn from the affinity pulldown experiments. The results confirmed that the phospho-SPTF peptide possesses a much stronger affinity (K\u003csub\u003ed\u003c/sub\u003e [95% CI]\u0026thinsp;=\u0026thinsp;206 nM) toward the BRCA1-tBRCT domain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec) than does the non-phosphorylated counterpart of this peptide (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). Interestingly, the phospho-mimetic EPTF peptide bound BRCA1-tBRCT with ~\u0026thinsp;30-fold weaker affinity, while DPTF peptide did not exhibit significant binding affinity, suggesting these mutations do not truly mimic phospho-Ser. As expected, peptides with Ser642 or Phe645 replaced with Ala also did not show significant affinity for BRCA1-tBRCT (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). Collectively, the results presented provide evidence that phosphorylation of SETX-Ser642 facilitates the formation of the SETX-BRCA1 complex.\u003c/p\u003e\n\u003cp\u003eFinally, to further corroborate the requirement of SETX-pSer642 for BRCA1-interaction, we generated SETX-S642A (SA) constructs for expression in insect cells and human cells. FLAG-co-IP from 293T cells co-expressing GFP, FLAG-tagged SETX-WT or SETX-SA, and Myc-tagged BRCA1 showed a significantly reduced interaction of SETX-SA with BRCA1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed). The breast cancer associated BRCA1 pathogenic variants S1665F (SF) and M1775R (MR) were reported to destablize the tBRCT domain and disrupt its interaction with the SxxF motif of BACH1/FANCJ, Abraxas, and CtIP\u003csup\u003e34\u0026ndash;37\u003c/sup\u003e. In BACH1/FANCJ, phospho-Ser990 of the phospho-SxxF motif forms hydrogen bonds with Ser1655 and Lys1702 of BRCA1, while the Phe993 residue fits in a hydrophobic pocket comprised of Phe1704, Met1775 and Leu1839 of BRCA1\u003csup\u003e36\u003c/sup\u003e. We co-expressed Myc-tagged BRCA1-WT, and the mutants, \u0026Delta;BRCT, SF or MR with GFP-FLAG-tagged SETX in 293T cells and performed co-IP studies, which showed that the BRCA1 mutants are impaired for SETX interaction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eEnhancement of SETX activity by BRCA1-BARD1\u003c/h2\u003e\n\u003cp\u003eHaving established direct interaction between SETX and BRCA1-BARD1, we next investigated possible functional synergy of these protein factors in R-loop resolution. Firstly, we examined BRCA1-BARD1 binding to R-loops \u003cem\u003ein vitro\u003c/em\u003e. Both BRCA1-BARD1 and BRCA1\u003csup\u003e\u0026Delta;BRCT\u003c/sup\u003e-BARD1 bound R-loops with comparable affinities (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea), indicating that deletion of the BRCA1-tBRCT domain does not significantly impact the R-loop binding activity of BRCA1-BARD1. However, SETX-mediated unwinding of telo R-loop was stimulated by BRCA1-BARD1, but not by BRCA1\u003csup\u003e\u0026Delta;BRCT\u003c/sup\u003e-BARD1 that does not interact with SETX (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). We note that BRCA1-BARD1, but not BRCA1\u003csup\u003e\u0026Delta;BRCT\u003c/sup\u003e-BARD1 could also stimulate unwinding of short synthetic R-loop via SETX (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e\n\u003cp\u003eNext, we asked whether the RAD52 protein (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec), which binds R-loops avidly (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed) and physically interacts with SETX \u003cem\u003ein vitro\u003c/em\u003e and in cells (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee,f), would affect the R-loop resolution activity of SETX. Importantly, we found that RAD52, at amounts stoichiometric to that of SETX, strongly inhibits R-loop resolution by SETX (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). In this analysis, we also included the human RAD52\u003csup\u003eK144A\u003c/sup\u003e mutant protein that is impaired for R-loop binding (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec,d) as previously reported\u003csup\u003e24\u003c/sup\u003e. The results showed that RAD52\u003csup\u003eK144A\u003c/sup\u003e failed to inhibit SETX mediated R-loop unwinding, thus providing direct evidence to link R-loop binding by RAD52 to its inhibitory action on SETX-mediated R-loop resolution. Importantly, we found that BRCA1-BARD1 helps alleviate the inhibitory effect of RAD52 on R-loop unwinding by SETX (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). These results highlight stimulatory effects of BRCA1-BARD1 on SETX\u0026rsquo;s R-loop unwinding activity and a potential mechanism for overcoming the inhibitory effects of R-loop binding factors such as RAD52.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRequirement of SETX S642 phosphorylation for R-loop resolution\u003c/strong\u003e \u003cstrong\u003ein cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next carried out cell-based studies to interrogate the physiological significance of the SETX-BRCA1-BARD1 axis of R-loop resolution. Firstly, we observed that HAP1 SETX-KO cells, as compared to isogenic wild type cells, are hypersensitive to camptothecin (CPT) treatment (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea,b) and accumulate R-loops as revealed by immunostaining with the S9.6 antibody (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec), which recognizes RNA-DNA hybrids, and by S9.6 slot blot analysis (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed). Importantly, we confirmed that treatment of RNase H1, but not with RNase III, RNase T1, and RNase A, reduces DNA:RNA hybrids detection by S9.6 antibody (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee). We also carried out SETX-S9.6 proximity ligation assay (PLA) to test whether SETX associates with R-loops in cells. The results revealed that SETX colocalizes with R-loops in the nucleolus and nucleoplasm, whereas the treatment of RNase H1, but not with other RNases, disrupts the interaction between SETX and R-loops (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef). Moreover, CPT treatment dramatically enhances the association of SETX with R-loops (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg).\u003c/p\u003e\n\u003cp\u003eTo understand the significance of SETX\u0026rsquo;s catalytic activity and Ser642 phosphorylation in R-loop resolution, we constructed HEK293A cell lines stably expressing GFP and FLAG-tagged SETX-WT, EA (catalytic null) and SA (Ser642 phosphorylation null) mutants for testing. PLA analysis showed that while both SETX-WT and SETX-EA colocalized with R-loops, SETX-SA failed to do so, indicative of a requirement of Ser642 phosphorylation for SETX recruitment to R-loops (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Next, we performed S9.6-slot blot analysis of cells after down-regulation of endogenous SETX via a 3\u0026prime;-UTR targeting siRNA (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eh). While the ectopic expression of SETX-WT suppressed R-loop accumulation, cells expressing the SETX EA or SA mutant exhibited R-loop levels comparable to SETX-depleted cells that harbored the empty vector (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb, Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eh).\u003c/p\u003e\n\u003cp\u003eTreatment of SETX-depleted cells with and without Olaparib, an inhibitor of polyADP ribose polymerases (PARPi), also significantly increased \u0026gamma;H2AX foci formation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec,d), suggesting SETX is needed for the proper response to increased replication stress and avoidance of DNA double-strand breaks (DSBs) upon occurrence of such stress. Importantly, while expression of SETX-WT in these cells attenuated the level of \u0026gamma;H2AX foci, neither the SETX-EA nor SETX-SA mutant was able to do so (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec,d), indicating that SETX catalytic activity and phospho-Ser642 are both important for preventing DSB formation linked to R-loop accumulation. Interestingly, we also found increased RAD52 foci in SETX depleted cells or when the SETX-EA or SETX-SA mutant was expressed in these cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee), suggesting that cells become more reliant on the RAD52-dependent repair mechanism\u003csup\u003e23,24\u003c/sup\u003e under these circumstances. SETX knockdown also reduced clonogenic survival of 293A cells upon olaparib treatment, and this phenotype could be corrected by ectopic expression of SETX-WT, but neither SETX-EA nor SETX-SA was effective in this regard (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eSETX recruitment and function at replication forks\u003c/h2\u003e\n\u003cp\u003eThe increased PARPi sensitivity of SETX depleted or mutant expressing cells suggests that SETX is required for protection of stressed replication forks. We used iPOND (isolation of proteins on nascent DNA) to examine possible recruitment of SETX to replication forks in HeLa cells. The results showed that association of SETX with replication forks becomes greatly enhanced upon treatment of cells with pladienolide-B (PB), an RNA splicing inhibitor that is known to induce R-loops (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, lane # 11\u0026ndash;12). As expected, we found that expression of RNase H1, which digests RNA in RNA-DNA hybrids, leads to attenuation of SETX recruitment to replication forks in PB-treated cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, lane # 13\u0026ndash;14). PB treatment also increased the level of \u0026gamma;H2AX in replicating DNA that could be mitigated by RNaseH1 expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, lane # 11\u0026ndash;14).\u003c/p\u003e\n\u003cp\u003eNext, we conducted iPOND analysis of cells in which BRCA1 could be depleted by doxycycline-inducible expression of shRNA against BRCA1\u003csup\u003e40\u003c/sup\u003e, which showed clearly that the recruitment of SETX to replication forks are dependent on BRCA1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, lane # 15\u0026ndash;18). We also utilized in situ analysis protein interaction at DNA replication forks (SIRF)\u003csup\u003e41\u003c/sup\u003e to show that SETX recruited to replicating DNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). While GFP-tagged SETX-WT and SETX-EA formed SIRF foci with EdU incorporated nascent DNA in S-phase cells, fewer SETX-SA foci were seen in these cells, indicating that Ser642 phosphorylation is important for SETX recruitment and/or retention at replication forks (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e\n\u003cp\u003eFurthermore, we examined the effect of SETX depletion with or without co-expression of SETX mutants on transcription-replication conflicts in cells via PLA of proliferating cell nuclear antigen (PCNA) and RNA polymerase II (RPII), following the rationale given in other studies \u003csup\u003e42,43\u003c/sup\u003e. Specifically, it has been shown that transcriptionally active RP II associates with nascent DNA via a transient interaction with PCNA, which enables the resumption of transcription upon passing of the DNA replication machinery\u003csup\u003e44\u003c/sup\u003e. Depletion of SETX increased PCNA-RPII foci that could be eliminated via treatment with RNase H1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec). Importantly, SETX-EA or SETX-SA expressing cells harbored a significantly larger number of PCNA-RPII foci compared to those expressing SETX-WT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec). Furthermore, we examined the effect of SETX depletion or mutations on DNA replication with SIRF assay that measured PCNA-EdU foci. SETX depletion led to reduced PCNA-EdU SIRF foci, which phenocopied the treatment of wild type cells with the replicative stressor hydroxyurea (HU) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed). Importantly, expression of SETX-WT, but not either SETX-EA or SETX-SA in cells depleted for endogenous SETX restored PCNA-EdU SIRF foci (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eAltogether, the data presented herein reveal that (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) R-loop accumulation leads to enhanced association of SETX with DNA replication forks, (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) recruitment of SETX to replication forks is dependent on its interaction with BRCA1 but its catalytic activity is dispensable, (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e) SETX is needed for the avoidance of replication-transcription conflicts, and (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e) SETX deficiency negatively impacts the progression of DNA replication forks, a premise that is supported by DNA fiber analysis as we present below.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eSETX is required for progression of replication forks\u003c/h2\u003e\n\u003cp\u003eWe interrogated how SETX depletion and SETX mutations affect DNA replication by DNA fiber analysis. SETX knockdown led to a significant decrease in the IdU/CIdU ratio (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea,b) and an increase in the ratio of stalled forks, indicating that SETX is needed for fork progression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec). In contrast, normal fork progression is restored in cells depleted for SETX but simultaneously expressing RNaseH1, providing evidence that fork stalling is a direct consequence of R-loop accumulation in the SETX deficient cells. Notably, ectopic expression of SETX-WT, but not SETX-EA or SETX-SA, fully alleviated the fork stalling phenotype induced by SETX ablation.\u003c/p\u003e\n\u003cp\u003eWe also wished to define the role of SETX in mitigating the effect of CPT-induced R-loops on DNA replication. For this purpose, we treated cells with 1 \u0026micro;M CPT for 30 min after CIdU labelling, followed by IdU labelling, and then performed DNA fiber analysis. As expected, fork progression (IdU/CIdU ratio in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed,e) and normal fork restart events (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef) were reduced in SETX knockdown cells. This aberrant phenotype could be overcome by expression of RNaseH1 or SETX-WT, but not by SETX-EA or SETX-SA.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCell-based investigations have implicated SETX in R-loop avoidance, but, thus far, the challenge of purifying SETX has precluded mechanistic dissection of its role in this crucial genome maintenance process. Importantly, we have succeeded in obtaining highly purified full-length human SETX via expression in insect cells, allowing us to demonstrate its R-loop and nucleic acid unwinding activities that likely reflect a direct role of SETX in R-loop resolution in vivo. Furthermore, our studies have provided the requisite platform for determining the mechanism by which BRCA1-BARD1 regulates SETX activity. Previous studies on Sen1 and the SETX helicase domain suggested that SETX unwinds R-loop via binding the 5\u0026prime;-end of RNA, as would be consistent with a role of SETX in transcription termination\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e. However, we have now shown that SETX binds and unwinds R-loop substrates without a 5\u0026prime; ssRNA overhang (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). From our biochemical analysis, we conclude that SETX unwind R-loops and RNA embedded within dsDNA efficiently without any requirement for an RNA overhang or displaced ssDNA as is encountered in the R-loop structure. Moreover, we demonstrate that somatic SETX point mutations identified in metastatic breast cancer patients, namely N2010I (NI) and H2197R (HR), in the helicase domain, engender a dramatic defect in catalytic activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed,e, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), suggesting an etiological linkage of SETX R-loop unwinding defect with tumorigenesis.\u003c/p\u003e \u003cp\u003eBRCA1 is recruited to transcription pause sites that have a high propensity of accumulating R-loops, where BRCA1 is thought to promote transcription termination via SETX\u003csup\u003e7\u003c/sup\u003e. Published studies have furnished evidence for a role of BRCA1 in R-loop resolution in specific genomic regions, and loss of BRCA1 lead to R-loop accumulation in breast tumors\u003csup\u003e30,45\u003c/sup\u003e. However, it has remained unclear whether BRCA1 directly interacts with SETX and if it participates in R-loop resolution with SETX. In this study, we have demonstrated a physical complex of SETX with BRCA1-BARD1 and that phosphorylation of the serine residue in the S\u003csup\u003e642\u003c/sup\u003ePTF motif of SETX enhances its interaction with the tBRCT domain of BRCA1. The SETX-S642A (SA) mutation and BRCA1-tBRCT pathogenic mutations S1655F (SF) and M1775R (MR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which reportedly destabilize the BRCA1-tBRCT domain\u003csup\u003e34\u0026ndash;37\u003c/sup\u003e, impair SETX-BRCA1 interaction. Even though the pathogenicity of BRCA1-SF and MR variants has been attributed to defective homology-directed DNA repair \u003csup\u003e46\u0026ndash;48\u003c/sup\u003e, our findings, together with a recent study demonstrating a role of the BRCA1-tBRCT domain in R-loop avoidance at rDNA loci\u003csup\u003e49\u003c/sup\u003e, implicate impaired R-loop resolution as a contributing factor to the pathogenicity incurred by the salient tBRCT mutations.\u003c/p\u003e \u003cp\u003eWe have leveraged the purified protein factors and \u003cem\u003ein vitro\u003c/em\u003e systems that we have developed to delineate the mechanistic underpinnings of the SETX-BRCA1-BARD1 axis in R-loop resolution. Firstly, our biochemical studies have shown that BRCA1-BARD1 binds R-loops with nanomolar affinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and stimulates R-loop unwinding by SETX (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Importantly, while both BRCA1-BARD1 and BRCA1\u003csup\u003eΔBRCT\u003c/sup\u003e-BARD1 bound R-loops with comparable affinities, the latter is not only defective in SETX stimulation, but is in fact inhibitory in this regard. Furthermore, we have found that RAD52, which also interacts with SETX (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) and binds R-loops with affinities comparable to BRCA1-BARD1 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), inhibits R-loop dissociation by SETX (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), further demonstrating specificity of the stimulatory effect of BRCA1-BARD1 on SETX. Moreover, we have shown that BRCA1-BARD1 alleviates the inhibitory effect of RAD52 on SETX R-loop unwinding (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Based on these new findings, we propose a model (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) where BRCA1-BARD1 (i) forms a physical complex with SETX and promotes SETX-mediated R-loop unwinding and (ii) helps overcome the inhibition imposed by RAD52. Based on this study and previous reports of RAD52 functioning in transcription-associated homologous recombination\u003csup\u003e23,24\u003c/sup\u003e, we speculate that RAD52-dependent repair comes into play when R-loops are converted into DSBs. In this regard, BRCA1-BARD1-dependent engagement and dissociation of R-loops help prevent RAD52 accumulation and DSB generation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In support of this hypothesis, we have shown that the SETX-SA phospho-null mutant that is impaired for BRCA1 interaction fails to be recruited to R-loops, and as a result, excessive R-loops (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b), RAD52 foci, and DSBs accumulate in cells expressing this mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,e). Interestingly, even though the catalytic null SETX-EA mutant remains robustly recruited to R-loops, cells expressing this mutant also exhibit R-loop accumulation as well as elevated RAD52 foci and DSBs. We note that cells depleted of SETX or expressing the EA or SA are also sensitized to PARPi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Interestingly, PARP1 is recruited to R-loops, and inhibition of PARP activity or its depletion leads to increased R-loop accumulation and genomic instability\u003csup\u003e50\u003c/sup\u003e. This could explain why PARP1 inhibition in cells lacking SETX activity induces synthetic lethality as we have shown here. As such, tumors harboring mutations that abrogate SETX activity or confer defects in SETX-BRCA1 interaction may be targeted via PARPi.\u003c/p\u003e \u003cp\u003eR-loops reportedly block replication machinery via head-on conflicts\u003csup\u003e42\u003c/sup\u003e, and lead to gaps in the nascent DNA strands via mechanisms suggested in other studies\u003csup\u003e51,52\u003c/sup\u003e. Even though yeast Sen1 protein has been reported to localize to replication forks to promote fork progression and maintain genome stability\u003csup\u003e21,53\u003c/sup\u003e, an analogous role of SETX has not been demonstrated in human cells. Importantly, our iPOND and SIRF-PLA analyses revealed the presence of SETX at replication forks in an R-loop dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,c). The requirement of BRCA1 and SETX-phospho-Ser642 for replication fork association of SETX provides formal evidence for the SETX-BRCA1-BARD1 ensemble in the prevention of transcription-replication conflicts via R-loop resolution. Accordingly, SETX knockdown cells experience elevated transcription-replication conflicts, a phenotype that can be complemented by ectopic expression of SETX-WT, but not the SETX-SA or SETX-EA mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Concordantly, mutant SETX cells experience elevated impediment of replication fork progression and increased fork stalling (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn summary, we have developed reconstituted systems to demonstrate that full length SETX has robust R-loop unwinding activity, physically interacts with BRCA1, and that BRCA1-BARD1 upregulates the R-loop unwinding activity of SETX. Altogether, these findings provide the molecular basis for elucidating the role of the SETX-BRCA1 axis in R-loop resolution and the promotion of replication fork procession in a transcribing genome, preventing generation of R-loop associated DSBs and replication stress.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Dr. Claus Azzalin at iMM,\u0026nbsp;Universidade de Lisboa, for providing the telo-R-loop template plasmid, Dr. Lee Zou at Duke University for RAD52\u003csup\u003eK144A\u003c/sup\u003e bacterial expression plasmid, and Dr. Bing Xia at Rutgers University for BRCA1-WT, ∆BRCT, S1665F and M1775R expression plasmids. We thank Dr. Hardeep Kaur (P.S. lab) for sharing important reagents. AD was supported by a Brown-Coxe postdoctoral fellowship from Yale University, a Jean B Kempner postdoctoral fellowship from University of Texas Medical Branch at Galveston, TX, and Office of Postdoctoral Affairs Fellowship from UT Health San Antonio, TX. This study was supported by National Institutes of Health grants RO1 ES007061, R35 CA241801, PO1 CA092584 (P.S.), RO1 CA168635 (G.M.K. and P.S.), R01GM141091 and R01CA268641 (W.Z.), U19AI150574, R01CA219836 (D.Z.), R50 CA265315 (Y.K.), P30 CA054174 (P.S., S.K.O. and D.Z.), R01 CA246807 (S.B.), R01 CA205224 (R.H.), R01 GM136717, R01 CA237286 (A.V.M.), R01CA152063 1R01CA241554 (A.J.R.B.), and PO1 CA092584 (P.S., A.V.C., D.T.M. and M.S.T.), by awards from the Cancer Prevention and Research Institute of Texas (CPRIT) REI RR210023 (A.V.M.), RP150445 (A.J.R.B.), RP210102 (W.Z.), and RP220269 (R.H.), by an award from the Congressionally Directed Medical Research Programs BC191160 (A.V.M.), by a Hyundai Hope on Wheels Scholar Grant (G.M.K.), by an American Cancer Society Research Scholar grant RSG-22-721675-01-DMC (W.Z.), and by a SU2C-CRUK award RT6187 (A.J.R.B.). The MST assays were carried out in the Center for Innovative Drug Discovery supported by CPRIT Core Facility Award RP210208 (D.Z.). P.S. is the holder of the Robert A. Welch Distinguished Chair in Chemistry (AQ-0012) and A.V.M. is the holder of the Joe R. and Teresa Lozano Long Chair in Cancer Research. The phosphopeptide mapping analysis was performed in the George L. Wright Center for Biomedical Proteomics supported by the Hampton Roads Biomedical Research Consortium. OJS holds the Anthem Distinguished Professorship in Cancer Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.D. generated and purified full length human SETX WT, point mutants and truncated proteins, BRCA1-BARD1 WT and truncated proteins, and RAD52\u003csup\u003eK144A\u003c/sup\u003e, designed and performed all biochemical assays, \u003cem\u003ein vitro\u003c/em\u003e pulldowns, and co-IP experiments. Y.K. purified human RPA and RAD52. J.H.J. performed the cell-based experiment including generation of stable cell lines, PLA, co-IP, S9.6 slot blot, foci formation experiments and clonogenic survival assay. Q.F., E.D. and B.D.L.P.A. performed the DNA fiber experiments. S.Z., supervised by D.Z. performed the MST experiments. F.L. performed the iPOND experiments. J.O.N. and O.J.S. were responsible for design and implementation of the mass-spectrometry-based studies. D.T.M., A.V.C. and M.S.T. generated BacMam-SETX and obtained affinity purified SETX from human cells for mass-spectrometric studies. A.B.T. provided insights on structural information on SETX Alphafold structure. W.L. generated HeLa-shBRCA1 cell line. A.O.H. performed some cell-based experiments. A.D., J.H.J., Y.K., G.K. and P.S. reviewed and participated in data interpretation together with other authors. A.D., W.Z., G.K. and P.S. conceived the project and developed experimental strategies. G.K., W.Z., and P.S. supervised and provided funding support. A.D. and P.S. wrote the manuscript with input from all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eProtein Purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo express 6XHis-MBP-SETX-Flag, a 800 ml culture of \u003cem\u003eTrichoplusia ni\u003c/em\u003e (Tni) insect cells (1x10\u003csup\u003e6\u003c/sup\u003e cells/ml, Expression Systems) in ESF921 media (Expression Systems) was infected with 8 ml baculoviral suspension followed by a 70 h incubation. Cells were collected by centrifugation and stored at -80\u0026deg;C until use. All the protein purification steps were carried out at 0-4\u0026deg;C. Cell pellet was resuspended in buffer L1 (50 ml per gram of pellet), composed of 50\u0026thinsp;mM Tris-HCl, pH 7.5, 500\u0026thinsp;mM KCl, 10% glycerol, 1 mM EDTA, 1\u0026thinsp;mM DTT, 0.01% NP-40, 2 mM ATP, 4 mM MgCl\u003csub\u003e2\u003c/sub\u003e, cOmplete protease inhibitor cocktail (MilliporeSigma), and 1 mM PMSF, with sonication. The lysate was clarified by ultracentrifugation at 100,000 x g for 45 min before being incubated with 1 ml Anti-FLAG-M2 affinity resin (MilliporeSigma) for 1 h. The resin was washed with 200 ml buffer W (50\u0026thinsp;mM Tris-HCl, pH 7.5, 500\u0026thinsp;mM KCl, 10% glycerol, 1 mM EDTA, 1\u0026thinsp;mM DTT, 0.01% NP-40, 2 mM ATP, 4 mM MgCl\u003csub\u003e2\u003c/sub\u003e) and then with 50 ml buffer T1 (50\u0026thinsp;mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1\u0026thinsp;mM DTT, 0.01% NP-40) with 100\u0026thinsp;mM KCl. SETX was eluted with 10 ml buffer T1 with 100\u0026thinsp;mM KCl, 0.25 mg/ml Flag peptide (Sigma) and cOmplete protease inhibitor cocktail (MilliporeSigma). The elution was fractionated in a HiTrap Q HP (1 ml) column (Cytiva) with a 30 ml gradient of 100-500 mM KCl in buffer T1. Peak fractions were concentrated using Amicon\u0026reg; Ultra Centrifugal Filters (100 kDa MWCO), divided into 2 \u0026micro;l aliquots, flash frozen in liquid nitrogen and stored at -80\u0026ordm;C. SETX mutants and truncated proteins were purified using the same procedure.\u003c/p\u003e\n\u003cp\u003eA BRCA1(Flag)-BARD1(6XHis) MacroBac plasmid was generated via ligation independent cloning in 438a vector and recombinant baculovirus was generated in Sf9 insect cells. BRCA1-BARD1 was expressed in 800 ml Tni cells (1x10\u003csup\u003e6\u003c/sup\u003e cells/ml) and purified following our established method \u003csup\u003e33\u003c/sup\u003e. BRCA1\u003csup\u003eD\u003c/sup\u003e\u003csup\u003eBRCT\u003c/sup\u003e-BARD1 was expressed and purified following the procedures developed for its wild type counterpart.\u003c/p\u003e\n\u003cp\u003eGST-tagged BRCA1-tBRCT (residue 1528-1863) and BARD1-tBRCT (residue 423-777) were expressed in \u003cem\u003eE coli\u003c/em\u003e BL21 Rosetta cells transformed with pGEX6p1-GST-BRCA1-tBRCT or pGEX6p1-GST-BARD1-tBRCT. Cells were grown in 6L cultures until OD 0.7 then protein expression was induced by the addition of 0.2 mM IPTG followed by a 18 h incubation at 16\u0026deg;C. All the subsequent steps were carried out at 0-4\u003csup\u003eo\u003c/sup\u003eC. Cells were harvested by centrifugation, resuspended in 50 ml buffer T2 (50\u0026thinsp;mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1\u0026thinsp;mM DTT, 0.1% NP-40) with 500\u0026thinsp;mM KCl, 2 mM PMSF, and cOmplete protease inhibitor cocktail), and lysed by sonicated. Clarified cell lysate was incubated with 1 ml Glutathione-Sepharose resin (Cytiva) for 1 h at 4\u0026deg;C, followed by washing with 200 ml buffer T2 with 50 mM KCl, 2 mM ATP, and 4 mM MgCl\u003csub\u003e2\u003c/sub\u003e. Then protein was released from the resin by cleaving the GST tag in an overnight incubation with 5 ml HRV-3C protease (Pierce) at 4\u0026deg;C. For obtaining GST-tagged proteins, elution was performed using buffer T2 with 500\u0026thinsp;mM KCl,10 mM glutathione, and cOmplete protease inhibitor. Proteins were further purified in HiTrapSP-HP with a gradient of 100-500 mM KCl in buffer T2, followed by size exclusion chromatography in a Superdex 200 column (Cytiva) in buffer T2 with 200 mM KCl. Peak fractions were concentrated, divided into 5 \u0026micro;l aliquots, flash frozen and stored at -80\u0026ordm;C.\u003c/p\u003e\n\u003cp\u003eRAD52 (WT), RAD52\u003csup\u003eK144A\u003c/sup\u003e \u003csup\u003e56\u003c/sup\u003e, and \u003cem\u003eS. cerevisiae\u003c/em\u003e Rad52 were purified following our published procedures \u003csup\u003e57-59\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of R-loop and other nucleic acid substrates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the construction of nucleic acid substrates, PAGE purified oligonucleotides (IDT) (Supplementary Tables 1 and 2), were labeled at their 5'-terminus with T4-Polynucleotide kinase (NEB) using \u0026gamma;-\u003csup\u003e32\u003c/sup\u003eP-ATP. Labelled oligonucleotides were annealed with an equimolar amount of unlabeled oligonucleotides (Supplementary Table 2) by gradually decreasing the reaction temperature from 95\u0026ordm;C to 4\u0026ordm;C in a thermal cycler.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATP hydrolysis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 50 \u0026micro;l stock ATP solution that contained 1 \u0026micro;l \u0026gamma;-\u003csup\u003e32\u003c/sup\u003eP ATP (10 \u0026micro;Ci/\u0026micro;l, Perkin Elmer) and 100 \u0026micro;M of cold ATP was prepared. SETX-WT or mutant was incubated in 10 \u0026micro;l (final volume) buffer R1 (10 mM Tris-HCl, pH 7.5, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 50 mM NaCl, 1 mM DTT, 1% glycerol) with 1 \u0026micro;l of the \u0026gamma;-\u003csup\u003e32\u003c/sup\u003eP ATP mix without or with 1 \u0026micro;M ssDNA (D7) or ssRNA (R1) at 37\u0026deg;C. The reaction was halted by mixing 2 \u0026micro;l of the reaction mixture and 2 \u0026micro;l of buffer S (1% SDS and 20 mM EDTA), and the level of ATP hydrolysis was determined by phosphorimaging analysis after thin layer chromatography in PEI-cellulose sheet (Sigma\u0026ndash;Aldrich, Cat No. Z740237\u0026ndash;25EA; \u003csup\u003e60\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eR-loop binding assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe indicated protein species was incubated with \u003csup\u003e32\u003c/sup\u003eP-labelled R-loop in 10 \u0026micro;l buffer R2 (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1% glycerol, and 100 \u0026micro;g/\u0026micro;l BSA) for 30 min on ice. After adding 2 \u0026micro;l buffer LD (100 mM Tris\u0026ndash;HCl, pH 7.5, 10 mM EDTA, 50% Glycerol, 0.15% Orange G), EMSA was carried out on 5% polyacrylamide gels in TAE buffer (30 mM Tris-acetate, pH 7.4 and 0.5 mM EDTA). The gels were dried onto Hybond-N membrane on top of Whatman filter paper and then subject to phosphorimaging analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNucleic acid unwinding assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSETX-WT or mutant was incubated with 2 nM of the indicated \u003csup\u003e32\u003c/sup\u003eP-labelled substrate in 10 \u0026micro;l buffer R3 (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 2.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1.5 mM ATP, 10% glycerol, 0.2 \u0026micro;g/\u0026micro;l BSA, and 1 mM DTT), and 1 \u0026micro;l Rnasein (Promega; for RNA-DNA or R-loops) for 20 min at 37\u0026deg;C. The reactions were stopped by adding 1 \u0026micro;l 0.5% SDS and 1 \u0026micro;l proteinase-K (5 mg/ml) and a 5-min incubation at 37\u0026deg;C. After adding 2 \u0026micro;l buffer LD, reaction mixtures were resolved in 10% TAE native gels at 10\u0026deg;C. Gels were dried onto Hybond-N membrane on top of Whatman filter paper and then subject to phosphorimaging analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of plasmid telo-R-loop and unwinding assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTelo R-loop was generated via in vitro transcription following a protocol modified from the literature \u003csup\u003e29\u003c/sup\u003e. Briefly, 2 \u0026micro;g pcDNA6-Telo plasmid containing a\u0026thinsp;~\u0026thinsp;1 kb human telomeric repeat sequence was incubated with 2 \u0026micro;l T7 RNA pol (NEB) and ATP, CTP, GTP (2.5 mM each), 825 nM \u0026alpha;-P\u003csup\u003e32\u003c/sup\u003e-UTP (3000 Ci/mmol; PerkinElmer), and 2 \u0026micro;l RNasein (Promega) in 1X T7 RNA pol buffer (NEB) for 30 min at 37\u0026deg;C. The reaction was stopped by adding cordycepin (5 mM) and, after a 10-min incubation, MgCl\u003csub\u003e2\u003c/sub\u003e (5 mM) was added to stabilize the R-loops. Telo-R-loop was purified with a Micro Spin S-400 column (Cytvia), aliquoted, and stored at -20\u0026deg;C. Telo-R-loop unwinding assay was conducted with 1 ng of the substrate and the indicated amount of SETX-WT or mutant in 10 \u0026micro;l of buffer R3 (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM ATP, 10% glycerol, 0.2 \u0026micro;g/\u0026micro;l BSA, 1 mM DTT, and 1 \u0026micro;l Rnasein) at 37\u0026deg;C for 20 min. The reactions were stopped by adding 1 \u0026micro;l each of 0.5% SDS and proteinase-K (5 mg/ml) followed by a 5-min incubation at 37\u0026deg;C. Reaction mixtures were resolved on 0.9% agarose gels in TAE buffer. Gels were dried on Hybond-N membrane on top of Whatman filter paper and then subject to phosphorimaging analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffinity pulldown assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor MBP pulldown experiments, 1 \u0026micro;M MBP-tagged SETX or SETX\u003csup\u003e\u0026Delta;HD\u003c/sup\u003e was incubated with 1.5 \u0026micro;M BRCA1-BARD1 or BRCA\u003csup\u003e\u0026Delta;BRCT\u003c/sup\u003e-BARD1 at 4\u0026deg;C for 30 min in 20 \u0026micro;l buffer M (25 mM Tris-HCl pH 7.5, 10% Glycerol, 0.5 mM EDTA, 0.05% Igepal CA-630, 1 mM DTT, and 150 mM KCl), with protein complexes being captured with 20 \u0026micro;l amylose resin (NEB). For GST pulldown experiments, 5 \u0026micro;M GST-tagged BRCA1-BRCT or BARD1-BRCT was incubated with 3 \u0026micro;M SETX\u003csup\u003e\u0026Delta;HD\u003c/sup\u003e or SETX-HD at 4\u0026deg;C for 30 min in 20 \u0026micro;l buffer M, with protein complexes being captured with 20 \u0026micro;l glutathione sepharose 4 fast flow resin (Cytvia). The resin was washed three times with 100 \u0026micro;l buffer W containing 200 mM KCl before eluting bound proteins with 1X Laemmli buffer and a 4-min incubation at 65\u0026deg;C. The supernatant and eluate were analyzed by SDS-PAGE and Coomassie staining or western blotting.\u003c/p\u003e\n\u003cp\u003eTo test interaction of FAM-labelled SETX phospho-peptide and FLAG-tagged BRCA1-BARD1, or GST-tagged BRCA1-BRCT and BARD1-BRCT, 0.5 \u0026micro;M peptide was mixed with 1 \u0026micro;M of the indicated protein in buffer R1 for 30 min at 4 \u003csup\u003eo\u003c/sup\u003eC. Then, protein complexes were captured using either anti-FLAG-M2 resin or glutathione sepharose 4 fast flow resin by constant mixing for 30 min. After washing the resin three times with 100 \u0026micro;l buffer M containing 200 mM KCl, bound proteins were eluted 20 \u0026micro;l Laemmli buffer at 65\u0026deg;C for 4 min. The supernatant and eluate, 1 \u0026micro;l each, were spotted onto nitrocellulose membrane and imaged in Bio-Rad fluorescence imager. The same samples were also run on 7.5% protein gel and subjected to Western blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscale thermophoresis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePurified BRCA1-tBRCT was serially diluted 1:1 with HEPES buffer saline (pH 7.5) up to 12 times in a 384-well plate, starting from the highest concentration available (final concentration: 6.8 \u0026micro;M \u0026ndash; 3.3 nM). Following the addition of FAM-labeled SETX peptides (final concentration 10 nM for SPTF, APTF and SPTA; 20 nM for pSPTF, EPTF and DPTF), samples were loaded into regular glass capillaries (MO-AK002, Nanotemper Technologies GmbH, M\u0026uuml;nchen, Germany). Microscale thermophoresis was then measured using a Nanotemper NT. Automated (Nanotemper Technologies GmbH, M\u0026uuml;nchen, Germany). Fluorescent power was determined using the autodetection initially and set to 67%, 100%, 99%, 100%, 58%, 63% for SPTF, pSPTF, APTF, SPTA, EPTF and DPTF respectively, and IR laser power was set to medium level to apply the heat gradient to each capillary and light emitting diode power to track fluorescence migration along the heat gradient. Relative fluorescent data between 1.5s to 2.5s time window after IR laser onset was used for binding affinity evaluation. Data from 3 independent experiments (9 measurements for pSPTF) were fitted using the built-in models in the MO Affinity Analysis V2.3 software. The final data were plotted using GraphPad Prism 9.5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMass spectrometric analysis of SETX\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecombinant SETX was expressed in human Expi293F cells by infection with a BacMam baculovirus prepared from the pEZT-BM vector (Addgene #74099). The BacMam baculovirus was generated using the standard Bac-to-Bac procedures following manufacturer\u0026rsquo;s instructions (Thermo Fisher). For inducing phosphorylation of SETX, okadaic acid (Calbiochem) at 25 nM (final concentration) was added to cells at 44 h post infection and 1 \u0026micro;M CPT (Sigma) was added 1 h before cell harvesting. Cells were lysed in buffer L2 (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.3% NP-40, 10% glycerol) supplemented with 2 mM PMSF, 2 mM beta-mercaptoethanol, and protease inhibitors (ETDA-free protease inhibitor mini tablet (Millipore Roche), and Phosphatase inhibitor mini tablet (Thermo Fisher-Pierce)), with Dounce homogenization, followed by sonication. SETX-FLAG affinity purification was performed by incubating the clarified cell lysate with anti-Flag M2 resin (Sigma) at 4\u0026deg;C with gentle rocking for 1.5 h, followed by protein elution with 3x Flag peptide (APExBio) in buffer L2.\u003c/p\u003e\n\u003cp\u003eAffinity purified SETX from human cells were precipitated with acetone, and separated by SDS-PAGE followed by Coomassie Blue staining. SETX containing gel slices were excised, cut into small 1mm\u003csup\u003e2\u003c/sup\u003e pieces, washed in solution A (50 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e pH 8.0), destained in solution B (50 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e/50% acetonitrile), dehydrated in solution C (100% acetonitrile), and dried in a vacuum concentrator. Proteins were reduced with 10 mM DTT in 50 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e for 1 h at 56\u0026deg;C and alkylated with 55 mM iodoacetamide in 50 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e for 1 h at room temperature in the dark. Then, pulverized gel pieces were washed (with solutions A, B, and C), dried in a vacuum concentrator, and rehydrated with trypsin (Promega V511A, 1:20 enzyme:substrate ratio) in solution D (50 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e/10% acetonitrile) with a 30-min incubation on ice. Samples were covered with additional solution D and incubated overnight at 37\u0026deg;C. Peptides were extracted with 50% acetonitrile/0.1% formic acid. The digestion solution and peptide extracts were combined and dried in a vacuum concentrator. The pellet was resuspended in 0.1% formic acid and the peptide concentration determined with a NanoDrop One spectrophotometer (Thermo Fisher Scientific, 205 nm, Scopes method). The peptide concentration was normalized before LC-MS/MS.\u003c/p\u003e\n\u003cp\u003eMS spectra were searched using MaxQuant software (version 2.1.0) against a human Swiss-Prot database (20,291 entries, canonical database). The following variable modifications were used for the searches: oxidation of methionine, deamidation of asparagine and glutamine, acetylation of protein N-termini and phosphorylation of serine, threonine and tyrosine residues. Carbamidomethylation of cysteine was included as a fixed modification. Trypsin was included as the protease with a maximum of two missed cleavage sites. The mass accuracy tolerances were set at 20 ppm for the first search and 10 ppm for the main search with a minimum peptide length of 7 amino acids. Match between runs was enabled with a match window of 0.7 min and alignment time of 20 min. The label-free quantification (LFQ) algorithm was used to calculate of LFQ intensities, and the LFQ minimum ratio count was set to 2. False discovery rates (FDR) for peptide spectral matches (PSM) and protein identifications were set at 1% (0.01). Complementary database searches for peptide and protein identification were performed using Mascot (Matrix Science).\u003c/p\u003e\n\u003cp\u003eThe relative abundance of non-phosphorylated and phosphorylated S642 in Non-CPT treated and CPT treated cells was determined using the LFQ intensity values obtained from MaxQuant. In a first step, the sum of normalized intensities values of all identified peptides (modified and unmodified) that contain S642 (DMHCLEASS*PTFSK, DMHCLEASS*PTFSKEPMK, DMHCLEASS*PTFSKEPMKVQDSVLIK, KDMHCLEASS*PTFSK and KDMHCLEASS*PTFSKEPMK) was obtained. The relative abundance of S642 phosphorylation in the two samples was calculated by determining the sum and proportion of the intensities of the peptides containing phosphorylated S642 compared to the summed intensity of the modified and unmodified peptides.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human HAP1 SETX wild type and knock-out (KO) cells were purchase from Horizon Discovery (HZGHC001565c005) and maintained in IMDM with 10% (v/v) FBS (Gibco) at 37\u0026deg;C. The human HEK293, HEK 293A, and HeLa cell lines were cultured in DMEM with 10% FBS (Gibco) at 37\u0026deg;C. 293A cell lines with stable expression of GFP (N-terminal) and FLAG (C-terminal) fused SETX wild type and point mutants were generated in presence of puromycin (2 \u0026micro;g/ml) selection, then GFP-positive cells were sorted by flow cytometry (BD science).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation and Western blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293 or HEK 293A cells were lysed with NETN buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, 50 U/ml MNase, 50 U/ml Benzonase) plus protease and phosphatase inhibitor cocktails (GeneDepot). Co-immunoprecipitation (co-IP) was carried out by incubating cell lysates with M2 FLAG-agarose resin (Sigma) at 4\u0026deg;C for overnight, followed by washes (3X) with buffer W and elution with Laemmli buffer. Co-IP samples were subjected to SDS-PAGE with 4\u0026ndash;20% SDS-polyacrylamide gradient gels and immunoblotted with the indicated antibodies.8\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProximity ligation assay (PLA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze co-localization of SETX at R-loops and co-localization of PCNA and RNAPII at replication-transcription conflict sites, proximity ligation assay (PLA) was carried out as per manufacturer\u0026rsquo;s protocol (Duolink In Situ Starter kits; DUO92101; Sigma-Aldrich). Briefly, U2OS cells were cultured in glass bottom dishes (SKU#801002; NEST). Cells were transfected with GFP-SETX-FLAG WT/EA/SA or EV (Fig.\u0026nbsp;5a), and with an endogenous SETX-3\u0026rsquo; UTR-targeting siRNA (Fig.\u0026nbsp;6c), for 48 hrs, or cells were treated with camptothecin (CPT; 10 \u0026micro;M) for 1 hr (Extended data Fig.\u0026nbsp;6g). For analyze co-localization of GFP/S9.6 or SETX/S9.6, cells were washed with PBS and pre-permeabilized with 0.25% Triton X-100/PBS for 10 min and fixed with 2% formaldehyde/PBS for 10 min. For treatment of RNases, cells were washed with PBS and pre-permeabilized in 0.25% Triton X-100/PBS with RNase H (10 U), RNase III (10 U), RNase T1 (10 U), or RNase A (10 \u0026micro;g) for 30 min at 37\u0026deg;C, before being fixed with 2% formaldehyde/PBS for 10 min. Otherwise, for analyze co-localization of PCNA and RNAPII, cells were washed with PBS and fixed with 2% formaldehyde/PBS for 10 min and cells were incubated with 0.25% Triton X-100/PBS for 30 min. Cells were washed with PBS twice, then kept in PLA Duolink blocking buffer (1% BSA in PBS) for 1 h at 37\u0026deg;C. Next, to detect colocalization of GFP-SETX-FLAG WT/EA/SA and R-loops, the cells were incubated with anti-GFP (ab290; abcam) and anti-S9.6 (Cat# MABE1095; Millipore) antibodies in Duolink antibody dilution buffer at 4\u0026deg;C overnight. In situ PLA probes (anti-mouse plus and anti-rabbit minus) were diluted 1:5 in Duolink antibody diluent and incubated to detect anti-GFP (rabbit) and anti-S9.6 (mouse) antibodies for 1 hr at 37\u0026deg;C. After washing with buffer A (10 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween 20) three times for 5 min each, dishes were incubated with Duolink ligase at 37\u0026deg;C for 30 min and then washed with buffer A twice for 5 min each. Amplification mix prepared as per manufacturer\u0026rsquo;s instructions was added into each dish and incubated at 37\u0026deg;C for 100 min in the dark. Lastly, dishes were washed with buffer B (200 mM Tris, pH 7.5, 100 mM NaCl) three times for 5 min each and 0.01x buffer B once for 5 min. Doulink In Situ mounting solution with DAPI (4\u0026rsquo;,6-diamidino-2-phenylindole) was used to mount glass bottom dishes in PLA for nuclear staining and applied to an Olympus FV3000 confocal microscope. Intensity of foci was determined using the ImageJ (1.53a version; NIH) software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA/DNA hybrid slot blot assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine R-loop levels in HAP1-WT and HAP1-SETX-KO cells or in 293A cells stably expressing GFP and FLAG-tagged SETX-WT/EA/SA, where endogenous SETX was depleted with SETX 3\u0026prime; UTR targeting siRNA, cells were lyzed with DNAzol (Invitrogen), and genomic DNA (gDNA) was sheared with sonication (50% of power; 10/20 sec interval of ON/OFF, 3X).The concentration of gDNA sheared was measured by Nano-photometer (N60; IMPLEN). The indicated concentration of gDNA was analyzed by the slot blot assay using Minifold I dot blot apparatus (Schleicher \u0026amp; Schuell). gDNA was treated with RNase H (10 U; NEB, M0297S), RNase III (10 U), RNase T1 (10 U), or RNase A (10 \u0026micro;g) at 37\u0026deg;C for 1 h to remove RNA/DNA hybrids. The membrane was blocked for 1 h at room temperate in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) with 5% milk and incubated overnight with the S9.6 antibody (Millipore; MABE1095) at 1:1000 concentration. Double-stranded DNA (dsDNA) antibody (ab27156; abcam) was used as a loading control of gDNA. The signals were detected by Western blot and quantified using the ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells were transfected with transiently expressing GFP and FLAG-tagged SETX-WT/EA/SA and endogenous SETX-3\u0026rsquo; UTR targeting siRNA for 48 hrs. Cells were incubated with DMSO or PARP inhibitor (PARPi) for 4 h at 37C. Cells were fixed with 2% (v/v) formaldehyde in PBS for 15 min at room temperature and washed once with PBS and then, permeabilized with 0.5% Triton X-100 in PBS for 30 min. For R-loop detection via anti-S9.6 antibody in HAP1 SETX WT and KO cells, cells were pre-permeabilized with 0.25% Triton X-100 in PBS for 10 min, and then cells were fixed with 2% (v/v) formaldehyde in PBS for 15 min. Next, cells were blocked with 10% FBS/PBS for 20 min at room temperature and then treated with indicated antibodies in 10% FBS/0.25% Triton X-100 in PBS overnight at 4\u003csup\u003eo\u003c/sup\u003eC. Cells were washed twice with 10% FBS/PBS and incubated with secondary antibody for 1 h at room temperature. Cells were mounted in Vectashield medium with DAPI (Vector Labs). Images were captured in an Olympus FV3000 confocal microscope and processed with the ImageJ software or Olympus Cellsens software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClonogenic survival assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e293A cells stably expressing SETX-WT/EA/KA were transfected with SETX 3\u0026prime;-UTR targeting siRNA. After 2 days, cells were seeded at a density of 500 cells in 6-cm dishes. HAP1-WT and SETX-KO cells were seeded similarly. After being treated with CPT, cells were allowed to recover for 10 days. Colonies were fixed and stained with 0.5% crystal violet (C3886; Sigma). Viability was expressed as mean number of colonies obtained from three independent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eiPOND Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa control and shBRCA1 cells \u003csup\u003e40\u003c/sup\u003e were cultured in DMEM containing 10% FBS medium. For BRCA1 knockdown, the cells were treated with 1 \u0026micro;g/ml doxycycline (Clontech Cat. No. 631311) for 3 days. RNaseH1-GFP was expressed 24 h prior to treatment with 1 nM Pladienolide-B (PB). The iPOND assay was performed as described previously (Sirbu, et al., 2012). Briefly, 1x10\u003csup\u003e8\u003c/sup\u003e cells were labeled with 10 \u0026micro;M EdU for 15 min at 37\u0026deg;C and washed once with EdU-free media. Then cells were treated with 1 nM PB for 24 h to induce R-loops. Following cross-linking treatment with 1% formaldehyde/PBS for 25 min, cells were quenched with 0.125 M glycine and washed with PBS three times. Collected cells were resuspended in permeabilization buffer (0.25% Triton-X/PBS) and incubated at room temperature for 30 min, then washed once with cold 0.5% BSA/PBS and PBS. Cells were collected by centrifugation and resuspended in click reaction buffer (10 \u0026micro;M Biotin-azide, 10 mM Na ascorbate, 2 \u0026micro;M copper(II) sulfate) and incubated with rotation at room temperature for 2 h. Cells were washed once with cold 0.5% BSA/PBS and PBS and collected by centrifugation. Cells were resuspended in buffer L3 (1% SDS with 50 mM Tris\u0026ndash;HCl, pH 8.0) and a cocktail of protease inhibitors comprised of aprotinin, chymostatin, leupeptin, and pepstatin A) and subject to sonication. After centrifugation (16,000 x g for 10 min at room temperature), supernatant was collected, diluted with cold PBS to contain 0.5% SDS and 25 mM Tris-HCl (pH 7.5) and mixed with an equal volume of streptavidin-agarose resin for 20 h at 4\u003csup\u003eo\u003c/sup\u003eC. The resin was washed once with 1 ml of cold lysis buffer, and then once with 1 M NaCl and twice with cold lysis buffer. Proteins were eluted with an equal volume of 2X Laemmli buffer at 95\u0026deg;C for 25 min. Western blotting was performed to analyze the eluate with anti-BRCA1(07-434, Millipore), anti-SETX (NB100-57542, Novus), anti-Histone H3 (C-2, sc-374669, Santa Cruz), anti-H2A.X (938CT5.1.1, sc-517336, Santa Cruz), and anti-PCNA (PC10, Novus) antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn situ analysis of protein interaction at DNA replication forks (SIRF)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK 293 cells stably expressing of GFP-SETX-FLAG WT/EA/SA in log phase were plated on glass bottom dishes and pulsed with 125 \u0026micro;M EdU in DMEM for 8 min and fixed with 2% paraformaldehyde (PFA) in PBS (pH 7.4) for 15 min at room temperature. Where HU treatment is indicated, EdU containing media was removed, dishes were washed twice with PBS, and incubated with DMEM containing 4 mM HU for 4 h before fixation. Next, cells were permeabilized with 0.25% Triton X-100 in PBS for 10 min at room temperature. Dishes were washed twice with PBS for 5 min. Cells were incubated in click-reaction cocktail (2 mM copper sulfate, 10 \u0026micro;M biotin-azide and 100 mM sodium ascorbate in PBS) for 1 h at room temperature. To observe localization of SETX-GFP or PCNA at replication forks, GFP-biotin and PCNA-biotin PLA was performed (see PLA section in the Star Methods), with primary anti-GFP or anti-PCNA and anti-biotin antibodies, respectively. Anti-Cyclin A2 (ab137769; abcam) antibody was used for detection of S-phase positive cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA Fiber assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA fiber assay was performed as described \u003csup\u003e61\u003c/sup\u003e. Briefly, 293T cells stably expressing GFP and FLAG-tagged SETX-WT/EA/SA were sequentially pulse labelled with the nucleotide analogs CIdU (100 \u0026micro;M, Sigma-Aldrich, C6891) and IdU (100 \u0026micro;M, Sigma-Aldrich, I7125), according to the schematic in each Fig.. Where indicated, 48 h prior to pulse labelling, cells were transfected with pEGFPN1-RNAseH using the Fugene HD transfection reagent (Promega) and with control siRNA or that targeting SETX using Lipofectamine RNAiMAX (Invitrogen). After labelling, cells were collected and resuspended in Trypsin-EDTA (Sigma-Aldrich, T3924) at 3.5x10\u003csup\u003e5\u003c/sup\u003e cells per agarose plug. The preparation of agarose plugs, proteinase K treatment of plug, plug washing, and \u0026beta;-agarase treatment of melted plug and fiber combing were performed as described \u003csup\u003e61\u003c/sup\u003e. Immunodetection was carried out using rat anti-BrdU (BU1/75 (ICR1) antibody (Abcam, ab6326)) at a dilution of 1:20 for CldU detection, and mouse anti-BrdU (Clone B44) (BD Bioscience, 347580) at a dilution of 1:5 for IdU detection. Goat anti-rat 488 (Invitrogen, A11006) and donkey anti-mouse Cy3 (Invitrogen, A31570) were used as secondary antibodies at a dilution of 1:100. To assess replication fork restart, 1.2x10\u003csup\u003e6\u003c/sup\u003e cells were used per agarose plug. Cells were initially labeled with CIdU for 30 min, followed by treatment with 1 \u0026micro;M CPT for 30 min, and then labeled with IdU for an additional 30 min. Fibers were imaged in an Olympus Fluoview FV3000 Confocal Laser Scanning Microscope and quantification was with the ImageJ software. The number of DNA fibers were analyzed for each experimental set are indicated in the Fig. legends.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the biochemical experiments, band intensity was quantified using ImageQuant (Cytvia) and statistical analysis was conducted using the GraphPad PRISM software. Two-way ANOVA, unpaired Student\u0026rsquo;s t test and Welch\u0026rsquo;s unpaired t test were performed as indicated in the Fig. legends. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM or \u0026plusmn;\u0026thinsp;SD. P value of \u0026lt;\u0026thinsp;0.05 is considered statistically significant.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNiehrs, C. \u0026amp; Luke, B. 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DNA fiber combing protocol using in-house reagents and coverslips to analyze replication fork dynamics in mammalian cells. \u003cem\u003eSTAR Protoc\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 101371 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"R-loop, SETX, BRCA1-BARD1, phospho-SPTF motif, replication-transcription conflict","lastPublishedDoi":"10.21203/rs.3.rs-3833044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3833044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSenataxin (SETX), a putative RNA-DNA helicase, is recruited to transcription pause sites via the tumor suppressor BRCA1. Here, we define the mechanism by which SETX-BRCA1 resolves transcription-associated R-loops to prevent deleterious outcomes. Specifically, we show that SETX unwinds R-loops, and that the complex of BRCA1 and its obligatory partner BARD1 binds R-loops and stimulates R-loop unwinding by SETX. Importantly, BRCA1-BARD1 alleviates the inhibitory effect of RAD52 on SETX-mediated R-loop unwinding. We also demonstrate that phosphorylation of Ser642 in SETX promotes its interaction with BRCA1 via the tandem BRCT domain of the latter. Accordingly, mutations in the catalytic domain or Ser642 in SETX lead to R-loop accumulation, transcription-replication conflicts, replication fork stalling, and DNA double strand breaks in human cells. Our results thus establish the molecular basis for functional synergy between SETX and BRCA1-BARD1 in R-loop resolution and the mitigation of transcription-replication conflicts to preserve genome integrity.\u003c/p\u003e","manuscriptTitle":"Mechanism of SETX-BRCA1-BARD1 complex in resolution of R-loops and transcription-replication conflicts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-15 05:37:40","doi":"10.21203/rs.3.rs-3833044/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-structural-and-molecular-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nsmb","sideBox":"Learn more about [Nature Structural \u0026 Molecular Biology](http://www.nature.com/nsmb/)","snPcode":"","submissionUrl":"","title":"Nature Structural \u0026 Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"84fd8228-5e41-4ede-b8fb-1b68161c5671","owner":[],"postedDate":"January 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28103036,"name":"Biological sciences/Biochemistry"},{"id":28103037,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2026-04-01T07:28:46+00:00","versionOfRecord":{"articleIdentity":"rs-3833044","link":"https://doi.org/10.1038/s41594-026-01778-8","journal":{"identity":"nature-structural-and-molecular-biology","isVorOnly":false,"title":"Nature Structural \u0026 Molecular Biology"},"publishedOn":"2026-03-31 04:00:00","publishedOnDateReadable":"March 31st, 2026"},"versionCreatedAt":"2024-01-15 05:37:40","video":"","vorDoi":"10.1038/s41594-026-01778-8","vorDoiUrl":"https://doi.org/10.1038/s41594-026-01778-8","workflowStages":[]},"version":"v1","identity":"rs-3833044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3833044","identity":"rs-3833044","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}