{"paper_id":"cbc2e4e4-1e94-4bce-a5a1-0902ff79ccde","body_text":"1 \n2 \nOxidative stress triggers RNAPII arrest through 3 \nPARylation and DNA damage 4 \n5 \nQuentin A. Thomas 1, Liyang Wu 1, Emma Lesage 1, Henriette K. M. Iversen1, David 6 \nLópez Martínez1, Smaragda Kompocholi 1, Haiyue Liu 1, Nicolás Nieto Moreno 1, and 7 \nLea H. Gregersen1* 8 \n9 \n10 \n1Center for Gene expression, D epartment of Cellular and Molecular Medicine, University of 11 \nCopenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark 12 \n13 \n14 \n15 \n*Correspondence: leag@sund.ku.dk16 \n17 \nRunning title: Oxidative damage triggers RNAPII arrest through PARylation and DNA base 18 \nlesions 19 \n20 \n21 \nKEYWORDS Transcription, nascent transcription, RNA polymerase II (RNAPII), RNAPII 22 \nelongation, DNA damage, oxidative stress, transcription-coupled DNA repair 23 \n24 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 2 \nSummary  25 \n 26 \nUV or gamma irradiation, as well as certain chemicals, generate DNA damage that disrupts 27 \ntranscription through a variety of well -characterised mechanisms. In contrast, the 28 \ntranscriptional response to oxidative stress remains poorly understood. Here, we describe a 29 \nrapid and widespread shutdown of transcription following oxidative DNA base damage.  By 30 \nmonitoring RNAPII occupancy and elongation dynamics, we demonstrate that oxidative stress 31 \ntemporarily halts RNAPII pause release and arrests the progression of elongation complexes 32 \nwithin the gene body. We present evidence that this occurs in a unique and transient manner, 33 \ncharacterised by abrupt arrest of elongating RNAPII  dead in its tracks , followed by rapid 34 \ntranscriptional recovery as DNA lesions are repaired.  We find that the restriction of initiation 35 \nand early elongation complexes is regulated by PARylation, whereas recovery of RNAPII 36 \narrested within the gene body requires DNA repair mediated by the base excision repair (BER) 37 \nand single-strand break repair (SSBR) pathways. 38 \n 39 \n 40 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 3 \nIntroduction 41 \n 42 \nOur genome is continuously exposed to genotoxic insults causing DNA lesions and genom ic 43 \ninstability 1. Lesions originating from oxidative damage are among the most frequently 44 \nobserved in the germline  2 and accumulation of mutations from oxidative damage has been 45 \nassociated with aging, neurodegenerative diseases , and cancer 1,3.  Actively transcribed genes 46 \nare particularly vulnerable to genomic instability, and rapid repair within genes is therefore of 47 \nutmost importance to prevent deleterious long-term effects 4,5. DNA repair and transcription are 48 \ninevitably coupled as they work on the same template. To maintain fidelity of cellular 49 \nprocesses, cells frequently respond to genotoxic stress by silencing transcription, either locally 50 \nor globally 4,6-9. In fact, certain types of DNA lesions can serve as a physical roadblock to 51 \ntranscription, causing a complete halt to RNAPII elongation 7. For instance, UV-induced bulky 52 \nDNA lesions directly impede RNAPII progression and necessitate repair through the 53 \ntranscription-coupled nucleotide excision repair pathway (TC -NER) to facilitate restart of 54 \ntranscription 9-13.  55 \nIn contrast, the direct impact on transcription is less clear in the case of non-bulky DNA 56 \nlesions and single-stranded breaks (SSBs) frequently induced by oxidative damage. While it is 57 \nknown that exposure to oxidative stress results in a global repression of transcription 8,14,15, it 58 \nremains unknown if and how oxidative DNA lesions impact the transcription machinery in vivo. 59 \nLesions originating from oxidative stress include non-bulky single-base modification such as 60 \n8-oxoguanine (8OG), abasic sites, and SSBs. In fact, SSBs is the most frequent type of DNA 61 \nlesions, with an estimated occurrence of tens-of-thousands per cell per day 1,16. Data generated 62 \nin vitro  suggest that RNAPII is able to bypass 8OG  DNA lesions  17-19, while products 63 \noriginating from further oxidation impede RNAPII progression 20. In addition, SSBs generated 64 \neither as a direct result of oxidative damage, or as BER repair intermediates have been shown 65 \nto impair RNAPII elongation in vitro 21. However, transcription assays performed with nuclear 66 \nextracts from hydrogen peroxide -treated cells remained transcriptionally inactive even on an 67 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 4 \nundamaged DNA template, suggesting that DNA lesions by themselves may not be sufficient 68 \nto explain the global transcriptional repression observed following oxidative stress 8. 69 \nBesides physical impairment caused by the DNA lesion itself, the DNA damage 70 \nresponse induces a coordinated recruitment of factors involved in DNA repair  to the sites of 71 \ndamage, which can elicit either local or global repression of transcription 22,23. The recruitment 72 \nof DNA repair factors can be facilitated either directly by the DNA lesion itself, or through 73 \nrecognition of lesion -stalled RNAPII, such as in TC-NER 7,9. Interestingly, rather than being 74 \ncaused by the lesion itself, local transcriptional repression around sites of double-strand breaks 75 \n(DSBs) is dependent on DNA damage signalling involving ATM, DNAPK, and poly(ADP-76 \nribosyl)ation (PARylation)-mediated regulation of RNAPII close to the site of damage as well 77 \nas changes in the chromatin environment 24-28. PARylation also serves to recruit NELF -E to 78 \nRNAPII stalled at DSBs 28. In addition to DSB s, PARylation is also implicated in the 79 \nrecruitment of DNA repair factors to  SSB lesions. Here , PARP1-mediated PARylation 80 \nstimulates recruitment of X-ray repair cross -complementing group 1 (XRCC1)  as well as 81 \nadditional factors involved in SSB repair (SSBR) 23,29,30. Interestingly, the lack of XRCC1 leads 82 \nto a failure of transcription recovery following oxidative stress, suggested to involve PARP1 83 \n‘trapping’ on DNA , which would create a physical block consisting of an unresolved repair 84 \nintermediate rather than the lesion itself 15.  85 \nIn addition to transcriptional repression induced by DNA damage, it is also becoming 86 \nevident that cellular stress such as heat shock result s in widespread repression of transcription 87 \n31-35. Multiple mechanisms have been reported to be involved in transcriptional attenuation 88 \nfollowing heat shock, including promoter -proximal pausing of RNAPII 33, NELF condensate 89 \nformation 36, and premature transcript termination 37.  90 \nUsing a variety of genome -wide approaches to track the transcriptional response  in a 91 \ntemporally and spatially resolved manner , we find that transcriptional activity in response to 92 \noxidative stress is dynamically regulated through early RNAPII pause release, coordinated by 93 \nPARylation in combination with rapid and reversible arrest of RNAPII in the gene body, 94 \ncontrolled at the level of DNA damage repair.  95 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 5 \n 96 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 6 \nResults 97 \n 98 \nOxidative stress induces a rapid transcriptional shutdown 99 \nTo investigate the immediate transcriptional response to oxidation-induced stress and DNA 100 \ndamage, we treated cells with a brief, sublethal dose of either H2O2 or menadione, the latter of 101 \nwhich leads to intracellular ROS formation (Fig. 1a, Supplementary Fig. 1a-b). Using metabolic 102 \nlabelling of nascent RNA with 4-thiouridine (4sU) or 5-ethynyluridine (EU) in human cells, we 103 \ntracked the global levels of RNA synthesis during and upon treatment recovery (Fig. 1b-d and 104 \nSupplementary Fig. 1c-e). Both H2O2 and menadione led to a pronounced decrease in overall 105 \nRNA synthesis across several cell lines (Fig. 1b-d, Supplementary Fig. 1c). In the case of brief 106 \nH2O2 treatment, this effect was almost immediate, with a rapid recovery of transcription activity 107 \n(Fig. 1b-c). In contrast , as expected, menadione induced a somewhat milder and temporally 108 \ndelayed response in terms of repression of nascent transcription levels  (Fig. 1b-d). Similarly, 109 \nlower concentrations of  H2O2 (250 M) also resulted in a delayed reduction  of nascent 110 \ntranscriptional activity (Supplementary Fig. 1c). In contrast, a high H2O2 dose (10  mM) or 111 \nmenadione (2 mM) resulted in a failure to recover normal transcription levels, impaired cell 112 \ngrowth, and apoptosis (Supplementary Fig. 1d-f). 113 \nTo investigate the effects on transcription genome-wide, we performed TT chem-seq 38 114 \nusing yeast spike -ins as an internal normali sation control to accurately quantify  changes in 115 \nnascent transcription in a spatially resolved manner. In agreement with the global quantification 116 \nfrom dot blots and EU assays, a dramatic reduction in overall transcription activity throughout 117 \nthe entire gene body of protein-encoding genes was observed (Fig. 1f-h). This was the case both 118 \nfor menadione and for H2O2 (Fig. 1f-h). In the case of H2O2, 99.5 % of differentially expressed 119 \nprotein-encoding genes were downregulated (Supplementary Fig. 1g). At lower H2O2 120 \nconcentrations, transcriptional repression was strongest at the 30 min recovery timepoint, while 121 \na lower menadione concentration led to the strongest repression at 1 -hour recovery 122 \n(Supplementary Fig. 2a-c), which again aligns with the quantifications from dot blots and EU 123 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 7 \nassays (Fig. 1b-c). Classical stress-response genes such as the immediate early genes FOS and 124 \nEGR1 were also repressed upon H2O2 and menadione treatment and with kinetics mimicking 125 \nthe global repression (Supplementary Fig. 2d-e), highlighting that even genes later activated as 126 \npart of various stress-response pathways do not initially escape the transcriptional repression 127 \nfollowing oxidative stress.  128 \nTo investigate the possibility that  direct inhibition of RNAPII activity might occur 129 \nbecause of its oxidation, we assembled in vitro transcription elongation complexes with purified 130 \nRNAPII, pretreated them  with increasing concentrations of H 2O2 and performed in vitro  131 \ntranscription assays. Whereas the transcriptional activity of RNAPII elongation complexes was 132 \nreduced at extremely high concentrations of H2O2 (>50 mM ), we failed to  observe any 133 \nimpairment of RNAPII activity with lo wer ( <5-10 mM) H 2O2 concentrations, making it 134 \nunlikely that the lack of transcriptional activity in cells is due to direct RNAPII inactivation 135 \n(Supplementary Fig. 3a-b).  136 \nThe results above indicate that transcriptional activity is dramatically reduced 137 \nthroughout the gene body almost immediately after exposure to oxidative stress. The dramatic 138 \ndecrease in nascent transcription within gene bodies can either be due to (1) dissociation of 139 \nRNAPII elongation complexes from the DNA template, resulting in fewer polymerases in the 140 \ngene body, or (2) stalling or arrest of RNAPII resulting in disruption of RNA synthesis by those 141 \nRNAPII elongation complexes. Interestingly, t he reduction of transcription did not at first 142 \nglance appear to be caused by  changes in the level of DNA -associated RNAPII as both 143 \nhyperphosphorylated and unphosphorylated RNAPII levels in chromatin remained unchanged 144 \n(Fig. 1i). 145 \nTo more precisely track RNAPII occupancy, we used ELCAP -seq 39 which maps 146 \nRNAPII binding at nucleotide resolution (Fig. 1j). To ensure that all RNAPII populations were 147 \neffectively captured regardless of their CTD -phosphorylation status, we used cells expressing 148 \nFLAG-tagged RPB3 (Fig. 1j and Supplementary Fig. 3c). Short RNA fragments protected from 149 \nnuclease digestion were captured by FLAG-immunoprecipitation of total RNAPII followed by 150 \nsequencing (Supplementary Fig. 3c-e). Despite the fact that transcription activity was markedly 151 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 8 \ndown during the initial response,  the levels of RNAPII in the gene body remain ed largely 152 \nunchanged ( Fig. 1k). The only differences being a slight accumulation of early elongation 153 \ncomplexes close to the TSS ( Fig. 1k). Intriguingly, this suggests that in response to oxidative 154 \nstress, elongating RNAPII complex es within the gene body  are not dissociated but instead 155 \nrapidly arrested, in a global manner leading to an immediate transcription impairment.  156 \n 157 \nOxidative stress represses RNAPII elongation within the gene body  158 \nTo confirm that the transcriptional repression observed after oxidative stress is indeed due to 159 \nreduced transcription in the gene body, we assessed the effect of oxidative stress on gene body-160 \nassociated RNAPII  in the presence or absence of the CDK9 inhibitor 5,6-Dichloro-1-β-D-161 \nribofuranosylbenzimidazole (DRB) (Fig. 2a). In these experiments, RNAPII was first cleared 162 \nfrom the gene body by a treatment with DRB for 3.5 hours, which prevents promoter proximal 163 \npause release, while allowing all RNAPII to run until the 3’ end of genes and terminate. DRB 164 \ninhibition is reversible, enabling release of RNAPII complexes into the gene body upon DRB 165 \nwashout. After the 3.5 hours DRB incubation, RNAPII was allowed to enter the gene body by 166 \n15 min of unperturbed transcription (RNAPII release phase), after which cells were treated with 167 \nH2O2 alone, or in combination with fresh DRB, and incubated  for another 15 min (Fig. 2a). 168 \nThis results in the release of RNAPII complexes into the gene body, which are in all cases 169 \nallowed to run for a total of 30 min  (15 min unperturbed + 15 min with indicated treatments) . 170 \nTo investigate the effect of oxidation on  RNAPII complexes already in the gene body , we 171 \nfocused our analysis on long genes (> 90 kb).  In agreement with previous data showing that 172 \nRNAPII travels approximately 2 kb/min and with a maximum speed around 4 kb/min  38, we 173 \nobserved that the RNAPII wavefront progressed to around 60-90 kb into the gene body in cells 174 \nnot treated with H2O2 (Fig. 2b-e, blue tracks). By contrast, progression of the wave  front into 175 \nthe gene body was clearly reduced in response to a brief oxidative stress treatment (Fig. 2b-e, 176 \nred tracks ). Importantly, the wavefront reached a similar point in the gene whether new 177 \ntranscription was inhibited by DRB or not at the time of oxidative stress treatment ( Fig. 2b-e; 178 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 9 \nmost easily seen by comparing red tracks in d and e). This strongly indicates that H2O2 treatment 179 \nalso arrested RNAPII molecules that were already in the gene body.   180 \n 181 \nArrested RNAPII complexes are not subject to ubiquitination and subsequent 182 \ndegradation   183 \nA hallmark of RNAPII stalled at bulky UV lesions is polyubiquitination of the largest RNAPII 184 \nsubunit RPB1  and subsequent RNAPII degradation 10,11. Unlike the rapid transcriptional 185 \nrepression that occurs following oxidative damage, UV -induced transcriptional repression 186 \npeaks around 45 min to 3 hrs after the initial exposure (Supplementary Fig. 4a). To examine if 187 \nRNAPII becomes ubiquitinated following oxidative damage, we tested RPB1 188 \npolyubiquitination in a time -course-dependent manner using the DSK2 affinity enrichment 189 \nstrategy for polyubiquitinated proteins 40. Although we did observe low levels of RPB1 190 \npolyubiquitination, these are much lower than RPB1 polyubiquitination after UV irradiation 191 \n(Supplementary Fig. 4b). Notably, unlike the case with UV 10, we did not observe any decrease 192 \nin overall levels of hyperphosphorylated transcriptionally engaged RNAPII during either the 193 \noxidative stress-induced RNAPII arrest or recovery (Supplementary Fig. 4c).  194 \nUpon UV-irradiation, RNAPII stalls at bulky DNA lesions which serve as a roadblock 195 \nfor polymerase elongation 7. Accordingly, removal of bulky DNA lesions by the transcription-196 \ncoupled nucleotide excision repair (TC -NER) pathway is a prerequisite for transcriptional 197 \nrestart following UV 10,13,41. Interestingly, TC-NER has also been implicated in lesion repair in 198 \nresponse to oxidative stress 42-45. To investigate the effects of NER following oxidative stress, 199 \nwe tracked nascent transcription recovery in  XPC and CSB knockout cells, which lack GG -200 \nNER and TC -NER, respectively 7. Knockout of XPC or CSB had little or no effect on the 201 \ntranscriptional response (Supplementary Fig. 4d). This is in stark contrast to the transcriptional 202 \nrecovery after UV, which is completely CSB -dependent 10,12,41. We also failed to detect 203 \nrecruitment by IP–mass spectrometry of CSB, XPC and other DNA repair factors involved in 204 \nNER to RNAPII complexes following oxidative stress (Supplementary Table 1). Together with 205 \nthe lack of significant RPB1 ubiquitination observed following oxidative stress (Supplementary 206 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 10 \nFig. 4b), this indicates  that RNAPII arrest following oxidative damage is mechanistically 207 \ndistinct from RNAPII arrest observed as a consequence of UV-irradiation.  208 \n 209 \nGene body stalled RNAPII resumes transcription upon recovery from oxidative stress 210 \nThe data above indicated that in response to oxidative stress, elongating RNAPII complex es 211 \nare rapidly arrested, dead in their tracks, leading to a global, temporary shutdown of nascent 212 \ntranscription. To further explore the transcription dynamics during the recovery of 213 \ntranscription, we tracked nascent transcript ion activity over time immediately after oxidation 214 \n(Fig. 3a). In agreement with the overall transcription level s indicated by  dot blots and EU 215 \nassays, we observe d increasing restart of nascent RNAPI I transcription 30 min and 1 hour 216 \nfollowing H2O2 removal (Fig. 3a-b, Supplementary Fig. 5a). Interestingly, transcription activity 217 \nwas restored through the gene body at both time -points, with higher  transcription activity 218 \nrestoration towards the 3’end of long genes at the 1-hour timepoint (Fig. 3a-b, Supplementary 219 \nFig. 5b). This again indicates that even RNAPII elongation complexes arrested by oxidation 220 \ninside the gene body can restart transcription. This point is clearly illustrated by tracking 221 \nnascent transcription recovery for  individual long genes, such as FARS2 (>570 kb) (Fig. 3b). 222 \nAs stated previously, RNAPII elongation rate measurements  indicate that RNAPII travels at 223 \napproximately 2 kb/min and with a maximum speed around 4 kb/min 38. So, if new nascent 224 \ntranscription during the recovery period originated only from newly initiated RNAPII or 225 \nRNAPII at the promoter-proximal pause site, all transcriptional activity would be restricted to 226 \nthe first 60 -120 kb after 30 min recovery and 120 -240 kb after 1 hour recovery (Fig. 3b, 227 \nindicated by arrows). However, recovery of transcription activity was observed throughout the 228 \ngene already at 30 min  and 1 hour recovery (Fig. 3b, indicated by dashed boxes ), suggesting 229 \nthat RNAPII complexes indeed resume transcription from within the gene body  following 230 \noxidation-induced transcriptional arrest.  231 \nTo further rule out the possibility that newly initiated transcription is responsible  for 232 \nthe recovery of transcriptional activity in the gene body , we finally tracked recovery in the 233 \nabsence of new release of RNAPII into the gene body by treating cells with DRB during the 234 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 11 \nrecovery period (Fig. 3c). We thus found that overall transcription activity was restored even 235 \nwhen DRB was present during recovery from H2O2 treatment, clearly seen at the 30 min and 1 236 \nhour time points (Fig. 3d) before already transcribing polymerases have run off the gene which 237 \ntakes place during  longer DRB incubations  (Fig. 3d, lower panel s). Using TT chem-seq, we 238 \nfurther confirmed that RNAPII complexes indeed were able to restart transcription from within 239 \nthe gene body in the presence of DRB. Indeed, as expected, transcriptional recovery  for one 240 \nhour after oxidative stress was shifted towards the 3’end and absent in the 5’ end of genes  in 241 \nthe presence of DRB ( Fig. 3e, dashed purple line; Fig. 3f, lowest panel) , and obvious from 242 \nquantification of nascent transcription levels in the first 25 kb compared to nascent transcription 243 \nlevels in the last 25 kb of genes only (Supplementary Fig. 5b). At the same time, the most 244 \nprominent change in RNAPII occupancy was a slightly increased pausing index (indicating 245 \naccumulation of early RNAPII complexes)  measured with ELCAP -seq during the recovery 246 \nphase ( Supplementary Fig. 5c). This indicates that the response to oxidative damage is 247 \nfundamentally different from transcriptional repression induced by bulky DNA lesions  which 248 \nresults in RPB1 ubiquitination, subsequent RNAPII degradation and transcription restart as a 249 \n5’ to 3’ wave front originating from the TSS by transcription from newly initiated RNAPII 250 \ncomplexes 7,10,46-48.  251 \nTo investigate if RNAPII elongation  rates change during the recovery phase  across 252 \ngenes, we computed the elongation index as the ratio of active elongation (TTchem-seq signal) 253 \nand RNAPII occupancy (ELCAP-seq), which was previously used as a proxy for RNAPII speed 254 \n49,50. As expected, this indicated a strong decrease in the RNAPII elongation index during the 255 \nH2O2 treatment ( Supplementary Fig. 5d). As transcription resumes, the RNAPII elongation 256 \nindex in the gene body increase d, while the elongation index in the beginning of the gene 257 \ndecreased slightly at the 30 min time point ( Supplementary Fig. 5d). At 1 hour recovery this 258 \nwas even more pronounced, with the elongation index almost back to normal levels in the 3’end 259 \nof the gene body, while the elongation index for early gene body RNAPII complexes decreased 260 \neven further (Supplementary Fig. 5d).  The inverse behaviour of RNAPII elongation indices for 261 \nearly RNAPII complexes and gene body RNAPII indicate that early stalled RNAPII complexes 262 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 12 \nand gene body -arrested RNAPII complexes resume transcription differently during the 263 \nrecovery period (Supplementary Fig. 5c-d). This could either be due to distinct mechanisms 264 \ncontrolling release of early RNAPII elongation complexes and gene body-arrested RNAPII, or 265 \ndue to differently con figured RNAPII complexes (bound by different RNAPII associated 266 \nfactors) impacting elongation kinetics of RNAPII complexes differently depending on their 267 \nposition at the time of transcription block.  268 \n 269 \nNELF mediated pausing of RNAPII is altered during the oxidative stress response  270 \nTo investigate whether  RNAPII complexes are remodelled during the global transcriptional 271 \nrepression caused by oxidative stress, we investigated the RNAPII interactome via proteomic 272 \nanalysis of RNAPII immunoprecipitates. We used two approaches (1) capturing total RNAPII 273 \ncomplexes using a FLAG-tagged RPB3 cell line  that allows us to capture  all RNAPII 274 \ncomplexes regardless of the phosphorylation status of the CTD, and (2) specifically capturing 275 \ntranscriptionally engaged RNAPII using the monoclonal 4H8 antibody, which recognizes CTD 276 \nhyperphosphorylated RNAPII (Supplementary Fig. 6a). Notably, u sing both approaches we 277 \nobserved an increased enrichment of NELF bound RNAPII complexes  in H 2O2 treated cells 278 \n(Fig. 4a-b and Supplementary Fig. 6b, Supplementary Table 1 ). In addition, we found  an 279 \nincrease in 5’ capping components such as CMTR1 and RNGTT associated RNAPII complexes 280 \nupon treatment with H2O2 (Fig. 4a-b). This is in line with  data suggesting that recruitment of 281 \ncapping factors is dependent on NELF 51 and suggests increased early RNAPII pausing. We 282 \nconfirmed the increased interaction between RNAPII and the NELF complex by RNAPII IP-283 \nwestern analysi s (Fig. 4c). Interestingly, while NELF recruitment to RNAPII was initially 284 \nincreased, it decreased during the recovery period and after 1 hour and  2 hours of recovery, 285 \nNELF was less associated with RNAPII than even the untreated conditions (Fig. 4c). This trend 286 \nwas also observed in RNAPII IP proteomics both at 1 hour and 2 h ours recovery 287 \n(Supplementary Fig. 6b).  288 \nTo address the role of NELF -mediated RNAPII regulation during the oxidative stress 289 \nresponse, we carried out a detailed analysis of RNAPII occupancy . ELCAP -seq provides 290 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 13 \nnucleotide resolution of RNAPII binding and is therefore ideally suited to investigate RNAPII 291 \npromoter-proximal pausing. This uncovered an increase in promoter-proximal pausing at 30 292 \nmin after oxidative stress (Fig. 4d and Supplementary Fig. 6c). Interestingly, pausing after 30 293 \nmin recovery was primarily increased at the canonical pause site (20-50 bp from the TSS), also 294 \nknown as the 1st pause site of NELF-mediated pausing 51,52. However, at later time points ( 1 295 \nhour and 2 hours recovery), RNAPII occupancy at the canonical pause site decreased, with the 296 \nRNAPII occupancy shifting downstream to the ‘2nd pause site’ coinciding with the +1 297 \nnucleosome 51 (Fig. 4d-f). Tellingly, a similar shift from 1st to 2nd pause site has previously been 298 \ndescribed to occur after loss of NELF 51,52. To test if the increased association between NELF 299 \nand RNAPII correlates with the changes in promoter-proximal pausing, we plotted the relative 300 \nNELF binding (ascertained by NELFE ChIP-seq) after normalisation to RNAPII binding levels 301 \n(spike-in normalised RNAPII ChIP-seq) (Fig. 4f). During the initial repression after oxidative 302 \nstress, more NELF accumulated at the promoter-proximal pause site relative to RNAPII , 303 \nindicating active NELF recruitment, or impaired NELF release, at paused RNAPII complexes 304 \n(Fig. 4f). However, during recovery, less NELF was bound to promoter-proximal RNAPII, in 305 \nagreement with our IP results and the observed shift in RNAPII pausing from the 1st pause site 306 \nto the 2nd pause site (Fig. 4c-f).  307 \nWe wondered if the increased NELF binding to RNAPII during the initial phase of the 308 \noxidative stress response serves to restrict paused RNAPII elongation complexes from entering 309 \nproductive elongation. To test this, we generated NELFC degron cell lines enabling us to track 310 \nnascent transcription in response to oxidative stress in cells lacking the NELF complex  311 \n(Supplementary Fig. 6d). Treatment with dTAG led to depletion of NELFC within 1 hour 312 \n(Supplementary Fig. 6e). As previously reported  51, NELFC depletion also resulted in 313 \ndegradation of additional NELF subunits, such as NELFA  (Supplementary Fig. 6e). 314 \nIntriguingly, however, the overall levels of nascent transcription either during the immediate 315 \nresponse to H 2O2 or in the recovery period were unaffected by loss of NELF (Supplementary 316 \nFig. 6f). As NELF condensation formation has been suggested to drive transcriptional 317 \nrepression during the heat shock response 36, we checked nuclear localisation of NELF during 318 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 14 \nthe oxidative stress response. As expected from previous data by others, changes in the nuclear 319 \nlocation of NELF condensate formation was observed following heat shock, but no indications 320 \nof condensate formation following oxidative stress were observed (Supplementary Fig. 6g). 321 \nThus, the NELF depletion experiments indicate that NELF -mediated regulation is not 322 \nresponsible, nor required, for the widespread transcriptional repression observed upon oxidative 323 \nstress. Instead, we observe that the position of early RNAPII pausing  is affected during the 324 \noxidative stress response in a NELF-dependent manner; first accumulating at the 1st pause site 325 \n(increased NELF binding) followed by a shift towards the more downstream +1 nucleosome 326 \npause site (decreased NELF binding).  327 \n 328 \nPARylation governs early transcriptional recovery  329 \nAnother mean s of promoting RNAPII stalling in response to DNA damage is PARylation 330 \ncatalysed by poly(ADP-ribose) polymerase 1 (PARP1)  14. PARP1 activation leads to 331 \nPARylation of PARP1 itself (auto-PARylation) and other proteins which serve as scaffolds for 332 \nrecruitment of DNA repair factors at the site of DNA lesions 23,53.  To test if PARylation might 333 \nbe relevant for the transcriptional response to oxidative stress, we first measured the level of 334 \nADP-ribose on proteins in a time-dependent manner. In agreement with previous data 14, we 335 \nobserved a strong and rapid increase in PARylation during the initial oxidative stress response 336 \n(Fig. 5a, left). The highest levels were observed during the H 2O2 treatment itself, coinciding 337 \nwith the time transcriptional arrest (Fig. 5a, left). During the recovery phase PARylation levels 338 \nrapidly decreased and almost reached baseline levels at the 2 hours recovery timepoint (Fig. 5a, 339 \nleft). PARylation can be modulated by treatment with either PARP inhibitor (PARPi) or 340 \npoly(ADP-ribose) glycohydrolase inhibitor (PARGi) 54. As expected, treatment of cells with 341 \nPARPi prior to oxidative stress abolished PARylation (Fig. 5a, right) while pre-treatment with 342 \na PARG inhibitor (PARGi) resulted in sustained PARylation ( Fig. 5b). To test if PARylation 343 \nmight play a functional role during the oxidative stress response we first tested the overall levels 344 \nof nascent transcription in cells treated with either PARPi or PARGi prior to the induction of 345 \noxidative stress. Strikingly, PARPi led to faster recovery of overall transcription levels, while 346 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 15 \nsustained PARylation following treatment with PARGi  resulted in reduced transcriptional 347 \nrecovery (Fig. 5c and Supplementary Fig. 7a-b). To address how  PARylation affects nascent 348 \nRNAPII transcription in a genome-wide manner, we performed nascent transcriptome profiling 349 \nby TTchem-seq. Confirming the dot blot results, we first observed higher levels of overall 4sU-350 \nlabelled RNA at the 15 min timepoint in PARPi treated cells (Fig. 5d). Somewhat surprisingly, 351 \nthe TTchem-seq data revealed that PARPi led to increased release of early RNAPII elongation 352 \ncomplexes into the gene body at the 15 min recovery timepoint (Fig. 5e-f). The same trend of 353 \nincreased nascent transcription levels primarily within the first 40 kb upon PARPi pre-treatment 354 \nwas also clearly observed at a single gene level ( Fig. 5g) and across genes regardless of gene 355 \nlength (Fig. 5h). With an RNAPII elongation rate of 2-4 kb/min, RNAPII complexes originating 356 \nfrom newly initiated RNAPII complexes or released from the promoter proximal site would be 357 \nexpected to travel 30 -60 kb into the gene body at the 15 min timepoint, which corresponds 358 \nnicely to the region in which the  primary increase in RNAPII transcription al activity upon 359 \nPARPi treatment was observed (Fig. 5e-h). This suggests that PARylation after oxidative stress 360 \ninhibits either RNAPII initiation or  RNAPII promoter-proximal pausing, or both. Strikingly, 361 \nthe spatial effect of PARPi treatment on nascent transcription across the gene was sustained 362 \neven at the 1-hour recovery timepoint , d espite seeing similar overall levels of nascent 363 \ntranscription between PARPi and control-treated cells at this timepoint ( compare Fig. 5c and 364 \n5h), again indicating that the effect of PARylation is primarily restricted to early elongation.  365 \nThis differential effect indicates that the transient arrest of RNAPII further into the 366 \ngene body rel ies on other mechanisms  or additional factors be yond PARylation signalling.  367 \nIntriguingly, NuMA, a nuclear protein associated with the mitotic spindle, binds RNAPII in a 368 \nPARP1-dependent manner with repair proteins TDP1 and XRCC1 and has been suggested to 369 \nhelp promote oxidative break repair 55. In apparent agreement with the notion that PARylation 370 \npromotes NuMA binding 55, we indeed observed increased association of NuMA with RNAPII 371 \nimmediately after oxidative stress (Supplementary Table 1). Importantly, however, NuMA was 372 \nnot detected in RNAPII elongation complexes captured by IP of hyperphosphorylated RNAPII, 373 \nbut only in IPs capturing total RNAPII compl exes (Supplementary Table 1) . This is i n 374 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 16 \nagreement with the idea that NuMA associates primar ily with initiating RNAPII, and in line 375 \nwith the findings above that PARylation primarily affects this RNAPII population.  376 \n 377 \nLack of SSBR prevents transcriptional recovery 378 \nA key function of PARP activ ation in response to oxidative ly induced DNA damage  is to 379 \nfacilitate recruitment of  the molecular scaffold protein XRCC1 to the single-stranded DNA 380 \nbreaks (SSB) generated either as a direct consequence of oxidative damage or by incision by 381 \nDNA glycosylases such as OGG1 , promoting efficient  base excision repair (BER) 29,30. To 382 \ninvestigate the role of XRCC1 in the transcriptional response to oxidative stress, we generated 383 \nXRCC1 KOs (Supplementary Fig. 7c). In agreement with previous reports 15, XRCC1 KOs 384 \nshowed a clear defect in recovery of nascent transcription following oxidative stress (Fig. 6a-385 \nb). This effect was observed  even 2 and 4  hours after treatment, suggesting that XRCC1 is 386 \ncritical for transcriptional recovery following oxidative stress  (Fig. 6a-b). Using nascent 387 \ntranscriptomics, we found that lack of XRCC1 led to an almost complete failure of  RNAPII 388 \ntranscription restart throughout the gene body ( Fig. 6c). Compared to WT cells, XRCC1 KOs 389 \nfirst and foremost displayed much less transcription recovery towards the end of long genes at 390 \nthe 1-hour recovery time point ( Fig. 6c), indicative of failure to restart gene body -arrested 391 \nRNAPII complexes. Previous reports have indicated that XRCC1 is dependent on PARylation 392 \nfor its rapid recruitment onto oxidatively induced SSB 30. Noticeably, pre-treatment of XRCC1 393 \nKOs with PARPi res cued the delay in overall nascent transcription levels observed in the 394 \nXRCC1 KOs ( Fig. 6d). This suggests that PARylation serve s a role in the transcriptional 395 \nresponse beyond simply facilitating XRCC1 recruitment to sites of DNA damage.  396 \nTo address the transcriptional interplay between XRCC1 and active PARylation  397 \ngenome-wide, we performed nascent transcriptome profiling by TT chem-seq of XRCC1 KOs, 398 \nwith and without PARPi pre-treatment (Fig. 6e-h). Interestingly, when focusing on elongation 399 \nclose to the TSS, PARPi completely reverse d the effect of XRCC1 KOs at 1 hr recovery (Fig. 400 \n6e). Thus, PARPi treatment both in WT and XRCC1 KOs led to enhanced transcription activity 401 \nearly in the gene body, in accordance with the previous results showing PARylation-dependent 402 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 17 \nrelease of early RNAPII complexes. However, looking at metagene plots for long genes, it was 403 \nobvious that PARPi treatment could not fully reverse the transcription recovery  towards the 404 \n3’end of long genes even at the 1-hour recovery timepointc. 6f). Tellingly, PARPi treatment in 405 \nXRCC1 KO cells did not fully rescue the lack of nascent transcription recovery towards the end 406 \nof genes to the same levels as PARPi treated cells, indicating a PARylation-independent effect 407 \nof XRCC1 on gene body recovery (Fig. 6f). This is also evident from single gene examples,  408 \nwhere transcription recovery in the beginning of CERS6 is similar for PARPi treatment in WT 409 \nand XRCC1 KOs, while 3’end recovery is not fully rescued by PARPi treatment in XRCC1 KOs 410 \n(Fig. 6g). Looking specifically at the PARPi effect in XRCC1 KOs across gene s with ranked 411 \naccording to their lengths, we again see an effect of PARPi restricted primarily to the initial 40 412 \nkb region ( Fig. 6h). This suggest s that while PARPi  promoted release of early elongation 413 \ncomplexes can partially ‘ override’ the lack of transcription recovery in XRCC1 KOs through 414 \nrelease of early elongation complexes, PARPi treatment cannot fully overcome transcriptional 415 \ndefects further into the gene body (Fig. 6h). To investigate if the effect of XRCC1 might be due 416 \nto PARP ‘trapping’ on DNA as previously reported 56, we checked the levels of chromatin-417 \nassociated PARP1 protein in a time-dependent manner. However, we found that PARPi did not 418 \nled to increased chromatin levels of PARP1,  either in WT cells or in XRCC1 KOs 419 \n(Supplementary Fig. 7d), arguing that PARP trapping is unlikely to be the cause of the 420 \ntranscriptional phenotypes observed upon PARPi treatment in our experiment. 421 \n 422 \nLack of OGG1 activity slows transcriptional restart  423 \nOne of the most frequent o xidative damage-induced DNA lesions is the non-bulky 8-424 \noxoguanine (8OG), which is primarily recognised by the base excision repair (BER) pathway. 425 \nAlthough RNAPII has been shown to bypass 8OG lesions present in naked DNA templates 17-426 \n19, it is unknown how 8OG might impact the transcription machinery in vivo. To investigate the 427 \nrole of BER and whether BER is required for transcription recovery  we chemically inhibited 428 \nthe 8OG DNA glycosylase-1 (OGG1). Interestingly, OGG1 inhibition (OGG1i) also led to a  429 \nclear delay in the recovery of transcription levels (Fig. 7a-b), indicating that recovery of nascent 430 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 18 \ntranscription is dependent on the BER pathway. This was  again further confirmed by nascent 431 \ntranscriptome profiling by TTchem-seq, which showed that transcriptional restart within the gene 432 \nbody at 1 hour recovery was almost absent ( Fig. 7c-f). Intriguingly, although treatment with 433 \nOGG1i delayed restart of  RNAPII, the level of transcriptional activity at later time points 434 \nrecovered close to levels of untreated cells (Fig. 7a-b), suggesting that rather than preventing 435 \ntranscriptional restart completely, BER defects such as those inflicted by lack of OGG1 activity 436 \ndelays transcriptional restart. This contrasts with  the situation in XRCC1 KOs where 437 \ntranscription activity remained impaired even at 4 hours recovery (Fig. 6a-b). To investigate 438 \nhow BER defects influence the PARylation -driven release of early RNAPII complexes, we 439 \ncombined OGG1i with PARPi  pre-treatment. Firstly, we wanted to see if PARPi treatment 440 \ncould override the dramatic sustained OGG1i nascent transcription repression. Indeed, similarly 441 \nto the situation for XRCC1, PARPi pre -treatment result ed in recovery of overall nascent 442 \ntranscription levels starting already at the 15 min recovery time point ( Fig. 7d) which was 443 \nmaintained throughout the recovery period (Fig. 7d and Supplementary Fig. 7e). To determine 444 \nif the PARPi  effect was restricted to release of early RNAPII elongation complexes, we 445 \nperformed TT chem-seq and looked specifically at the nascent transcription recovery for long 446 \ngenes at the 1-hour timepoint. Like the situation for XRCC1, the added transcriptional activity 447 \nbrought about by PARPi pre-treatment in OGG1i treated cells was primarily restricted to early 448 \nelongation complexes ( Fig. 7e). However, when focussing also on the 3’end of long genes, 449 \nPARPi pre-treatment was able to overcome the OGG1i elongation block to a greater extent than 450 \nin XRCC1 KOs (Fig. 7f). Nevertheless, gene body transcription levels in the 3’end of long genes 451 \nwere still greatly reduced compared to WT conditions ( Fig. 7e). Something that is also very 452 \nevident from an individual gene example of the long gene PAM (Fig. 7g). The spatially 453 \nrestricted effect of PARPi ‘force d’ early RNAPII release  was further confirmed by heatmaps 454 \nlooking across both long and short genes (Fig. 7h). Thus, again PARPi treatment leads to release 455 \nof early RNAPII elongation complexes , an effect that can partially override the elongation 456 \nblock observed in cells defective for OGG1-mediated repair (Fig. 7e-h, Supplementary Fig. 7e). 457 \nThis again suggests that while PARylation restricts early elongation complexes, DNA repair is 458 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 19 \ncrucial for resumption of RNAPII transcription within the gene body. An effect which becomes 459 \nparticularly evident when looking at the 3’end of long genes ( Fig. 7f-h). Together, these data 460 \nthus indicate that, after oxidative damage, PARylation is important to restrict release of RNAPII 461 \nfrom promoter-proximal areas, while DNA repair is required to remove DNA lesions in the 462 \npath of RNAPII. 463 \n 464 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 20 \nDiscussion  465 \n 466 \nGenotoxic-induced transcriptional attenuation occurs in response to UV and gamma 467 \nirradiation, as well as oxidative damage 6-8,10. However, the transcriptional response varies 468 \nsignificantly depending on the nature of the damage. In contrast to previously described 469 \nmechanisms of transcriptional attenuation following UV -induced damage, we find that 470 \noxidative damage induces a rapid and distinct transcriptional response, characterised by abrupt 471 \narrest of RNAPII within the gene body. Even more unexpectedly, we find that these RNAPII 472 \ncomplexes restart transcription from their site of arrest. This is in contrast with the UV response, 473 \nwhere transcriptional recovery relies on new transcription initiation and proceeds in 5’ to 3’ 474 \nwavefront from the TSS 7,10,13,47,48. The difference most likely stems from the type of damage 475 \ninduced. While UV induced bulky DNA lesions act as roadblocks for RNAPII, and lesion 476 \nstalled RNAPII itself plays a central role in recruiting of repair factors such as CSB 5,9,13, this 477 \nis not the case following oxidative damage. Unlike the UV response, RNAPII is neither 478 \nubiquitinated and subsequently degraded, nor are NER factors such as XPC or CSB required 479 \nfor transcriptional restart. Indeed, high doses of hydrogen peroxide for which transcription fails 480 \nto recover within a few hours , have been associated with RNAPII ubiquitination 8,57. We 481 \ntherefore speculate that RNAPII ubiquitination following oxidative stress serves only  as a 482 \nsecondary response , facilitating removal of persistently stalled RNAPII from the DNA 483 \ntemplate. At lower doses of oxidative damage, RNAPII restart s from within the gene body , 484 \nenabling a less disruptive and faster transcriptional recovery. Compared to the UV response, 485 \nthe kinetics of RNAPII arrest and restart following oxidative damage are extremely rapid. 486 \nWhereas the UV -induced transcriptional repression is strongest a few hours after the initial 487 \nexposure and recovery occurs 6 -24 hours post-UV 13, oxidative damage-induced repression is 488 \nimmediate and recovery of overall transcription levels are restored within 1-2 hours of recovery. 489 \nWe suspect that th is rapid response results from arrested RNAPII complexes being able to 490 \nrestart from within the gene body, thereby bypassing the need to physically remove RNAPII 491 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 21 \ncomplexes from the DNA template. This aligns with our observation that RNAPII complexes 492 \narrested due to oxidative damage are differently configured in terms of their associated factors.  493 \nSimilarly to the heat shock response , we observed an accumulation of NELF -paused 494 \nRNAPII during the transcriptional arrest and early recovery phase 36. However, unlike the heat 495 \nshock response, oxidative stress did not result in NELF condensate formation, nor was the 496 \npresence of NELF required for the transcriptional repression 36. In addition to its global role in 497 \nheat shock-mediated transcriptional downregulation, NELF is involved in local transcriptional 498 \nsilencing through its recruitment to sites of DSBs in a PARylation-dependent manner 28. These 499 \nobservations led us to question whether NELF might be recruited to RNAPII within the gene 500 \nbody in response to oxidative stress. However, NELF ChIP -seq data excluded this possibility. 501 \nInstead we find that  NELF-mediated pausing is not per se required for the transcriptional 502 \nresponse although it is impacting the pause site of RNAPII, leading to a transition of RNAPII 503 \npausing from the 1 st pause site to the 2 nd pause site (+1 nucleosome)  51. These observations 504 \nagain emphasise how global transcriptional repression can be achived through different 505 \nmechanisms depending on the type of cellular stress and the nature of DNA damage induced.  506 \nBulky DNA lesions induced by UV are known to block RNAPII progression both in 507 \nvitro and in vivo, while non-bulky DNA les ions such as those typically induced by oxidative 508 \ndamage have generally been considered not to induce RNAPII stalling 17-19. SSBs however have 509 \nbeen reported t o stall RNAPII elongation in vitro 21. This is most likely not due to the DNA 510 \nbreak itself, but rather to the aberrant 3’ termini of the gap impeding RNAPII progression  58. 511 \nOur data strongly suggest that RNAPII gene body arrest is dependent on DNA damage, which 512 \nis rapidly resolved through BER and SSBR, involving the DNA glycosylase OGG1 and 513 \nXRCC1. XRCC1 serves as a scaffold protein to facilitate recruitment of DNA repair enzymes 514 \nto SSB either generated directly enabling SSBR or to SSB generated as BER intermediates 59.  515 \nConsequently, lack of XRCC1 leads to a delay in SSBR and is associated with neuronal 516 \ndysfunction and neurological disease s, highlighting its important role in the maintenance of 517 \ngenome stability 59,60. However, the molecular interplay between SSBR and transcription has 518 \nuntil now not been well -studied. Here we describe how  the lack of XRCC1 leads to strong 519 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 22 \ndefects in RNAPII gene body recovery consistent with persistent occurrence of SSB within 520 \ngenes. Accordingly, cells lacking XRCC1 completely fail to recover transcription even at 4 521 \nhours after hydrogen peroxide treatment, as a consequence of sustained transcriptional 522 \nrepression within genes. Similar to XRCC1, OGG1i treatment results in very dramatic and 523 \nprolonged gene body RNAPII arrest which is sustained at the 1 -hour recovery timepoint. In 524 \ncontrast to lack of XRCC1, OGG1i -treated cells do eventually recover nascent transcription 525 \nlevels at the 2–4-hour recovery timepoint. We suspect this difference is due to additional DNA 526 \nglycosylases such as NEIL1, NEIL2 and MUTYH 61,62. Indeed, NEIL1 and to a lesser extent 527 \nNEIL2 have been shown to function as backup BER glycosylases working both on 8OG and 528 \nfurther oxidation products such as hydantoin lesions upon OGG1 TH5487 inhibitor treatment 529 \nor OGG1 depletion 61. This also fits well with mice knockout phenotypes, where individual 530 \nBER glycosylase knockouts display only a moderate increase in mutation frequencies, while 531 \ndouble or triple knockouts, such as OGG1/MUTYH knockouts display strong phenotypes 532 \ncharacterised by high susceptibility to cancer and shortened lifespan 63. In addition, alternative 533 \nrepair pathways that might compensate for the lack of OGG1 -mediated repair, such as NER , 534 \ncould facilitate a secondary response in case of persistently stalled RNAPII complexes 44. In 535 \ncontrast, XRCC1 is required both for SSBR and downstream in the BER pathway to resolve 536 \nSSB intermediates 59. We speculate that this explains why lack of XRCC1 leads to sustained 537 \ntranscription repression, as lack of XRCC1 cannot easily compensated for by alternative repair 538 \npathways.  539 \nOxidative damage -induced transcriptional repression coincides with high levels of 540 \nPARylation which quickly decrease as transcription recovers. Sustained PARylation achieved 541 \nthrough PARG inhibition maintains transcriptional repression, highlighting the dynamic 542 \nregulation of the transcriptional response through PARylation. Traditionally , PARylation has 543 \nbeen considered as a recruitment signal for DNA repair factors and plays a crucial role in SSBR 544 \nthrough recruitment of XRCC1 23. Lack of XRCC1 has also been suggested to promote PARP 545 \n‘trapping’ on DNA leading to a transcription block through maintained PARP1 binding to sites 546 \nof DNA lesions 56. However, while we find that cells lacking XRCC1 fail to recover gene body 547 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 23 \ntranscription following oxidative stress, PARPi treatment exerts a milder effect on gene body 548 \ntranscription, suggesting that the presence of the oxidative DNA lesions themselves rather than 549 \nPARylation-induced effects are the primary cause of gene body RNAPII arrest. Instead, we find 550 \nthat PARylation controls the release of early RNAPII complexes from the TSS  already at the 551 \n15 min recovery timepoint. Notably, if we had relied solely on quantification of overall levels 552 \nof nascent transcription following PARPi treatment , we would have concluded that PARPi 553 \ntreatment completely reverses the transcription inhibition as early as 15 min recovery. 554 \nHowever, nascent transcriptomics clearly show that the effect of PARPi is more complex and 555 \nspatially restricted to early RNAPII elongation complexes. Intriguingly, the effect of PARP 556 \ninhibition of early RNAPII elongation complexes is independent of BER and SSBR DNA repair 557 \nfactors such as OGG1 or XRCC1, as we show that PARPi treatment ‘overrides’ continued 558 \ntranscriptional repression and promotes release of early elongation complexes even in cells 559 \nlacking XRCC1 or following treatment with OGG1i. Based on our results, we suggest a spatial 560 \nrole of PARP1 which has hitherto been mistakenly overlooked.  561 \nThe question remains h ow PARylation restricts early RNAPII elongation complexes . 562 \nBoth NELF and the pTEFb component CycT1 have been reported as direct PARylation targets 563 \n14,64. PARylation of NELF has been suggested to promote elongation 64, whereas CycT1 564 \nPARylation induced by MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) disrupts CycT1 phase 565 \nseparation, correlating with reduced RNAPII hyperphosphorylation in vitro 14. In the case of 566 \nCycT1, this would suggest an inhibitory effect on RNAPII pause release. However, it remains 567 \nunknown if MNNG induce s a spatially separated transcriptional response  like hydrogen 568 \nperoxide. Another possibility is that PARylation of proteins associated with early RNAPII 569 \ncomplexes signals recruitment of additional factors such as NuMA which has been suggested 570 \nto impact oxidative damage repair through a yet undefined mechanism 55. Thus, elucidation of 571 \nthe precise molecular mechanism of PARylation dependent restriction of early RNAPII 572 \nelongation remains an exciting topic for future research and will undoubtedly provide new 573 \ninsight into how genotoxic DNA such as that induced by oxidative stress elicit s a highly 574 \nregulated transcriptional response.  575 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 24 \nAs PARP inhibition is not able to prevent the initial repression of transcription, we 576 \nsuggest that the immediate and strong transcriptional attenuation reflects arrest of RNAPII by 577 \noxidative DNA lesions and RNAPII -blocking repair intermediates, occurring in a PARP -578 \nindependent manner. This fits well with our results showing that the immediate transcriptional 579 \nrepression dramatically impacts RNAPII gene body activity. This is analogous to the situation 580 \nfor UV-induced bulky lesions, where the DNA damage itself directly impacts the transcription 581 \nmachinery. However, due to the type of DNA lesion and consequently how repair and RNAPII 582 \nstalling are dealt with, the two types of genotoxic insults led to completely distinct outcomes in 583 \nterms of transcriptional response. These differences are manifested in both the kinetics of the 584 \ntranscriptional response and  factor dependencies , underscoring the unique nature of 585 \ntranscriptional regulation in response to oxidative damage. 586 \n 587 \nWhile the coupling between transcription and nucleotide excision repair (NER) is well 588 \nestablished, known as TC-NER, and relies on DNA damage recognition via RNAPII stalling 589 \nwithin actively transcribed genes, the interplay between BER and transcription remains largely 590 \nunexplored. A defining feature of TC -NER is its preferential repair of bulky DNA lesions on 591 \nthe transcribed strand of active genes 7,9,13. Our findings suggest that transcriptional recovery 592 \nfollowing oxidative damage is closely linked to DNA repair mediated by BER and SSBR, 593 \npointing to the possible existence of transcription -coupled BER (TC-BER) and transcription-594 \ncoupled SSBR (TC-SSBR). It has been hypothesised that TC-BER may operate analogously to 595 \nTC-NER, favoring repair within transcription units. While some studies support preferential 596 \nBER of oxidative lesions in the transcribed strand in reconstituted transcription assays 65, others 597 \nreport no strand bias in the repair of 8 -oxoguanine (8OG) lesions 66, leaving the mechanistic 598 \nbasis of such coupling unresolved.  Unlike TC-NER, we do not observe direct recruitment of 599 \nDNA repair factors to RNAPII. This absence may reflect a more transient interaction between 600 \nthe transcription machinery and repair complexes, or the inherently rapid kinetics of BER and 601 \nSSBR compared to TC -NER 13,59. This underscores the need for further investigation into the 602 \nnature and dynamics of transcription-coupled repair beyond NER. 603 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 25 \n 604 \nIn summary, our findings demonstrate that oxidative damage triggers a dramatic and 605 \nimmediate global repression of nascent transcription. This transcriptional attenuation is 606 \ncharacterised by increased stalling of early RNAPII complexes near the TSS, as well as RNAPII 607 \narrest within the gene body.  A distinctive feature of this response is that gene body –arrested 608 \nRNAPII can resume transcription from within the gene body itself, enabling rapid recovery. 609 \nAlthough NELF -mediated regulation influences promoter -proximal RNAPII pausing and 610 \npositioning, NELF is not essential for the transcriptional repression.  Strikingly, we find that 611 \nseparate mechanisms govern the transcriptional response of early RNAPII elongation 612 \ncomplexes and gene body–arrested RNAPII. Early RNAPII stalling is regulated by PARylation, 613 \nwhereas recovery within the gene body depends on DNA repair mediated by BER and SSBR, 614 \nsuggesting that oxidative DNA lesions in vivo  impede RNAPII progression.  Thus, our 615 \ntemporally and spatially resolved characteri sation of the nascent transcriptional response to 616 \noxidative damage reveals previously unrecognized layers of genotoxic-induced transcriptional 617 \nregulation. These mechanisms serve to transiently repress global transcription through RNAPII 618 \narrest, orchestrated by PARylation and DNA damage repair pathways. 619 \n  620 \n 621 \n 622 \n 623 \n 624 \n 625 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 26 \nAuthor contributions  626 \nL.H.G. and Q.A.T conceived the project. Q.A.T., L.H.G., L.W., E. L., H. K.M. I., S.K., D.L.M., 627 \nand N.N.M.  performed experiments.  Q.A.T performed main experiments. L.H.G performed 628 \nproof-of-concept experiments, assay establishment and initial TT chem-seq experiments. L.W. 629 \nassisted in generating NELFC degron cell line, performed NELF microscopy experiments as 630 \nwell as UV and heat shock experiments. E.L. performed NELF and RNAPII ChIP -seq 631 \nexperiments. H.K.M.I. performed RNAPII Dsk2 pull -outs. S.K. performed initial EU assays. 632 \nD.L.M performed in vitro RNAPII transcription assays. N.N.M. generated XPC knockout cell 633 \nline. Q.A.T performed bioinformatic analysis , with input from H.L. L.H.G. wrote the 634 \nmanuscript with input from Q.A.T . All authors read and approved the final version of the 635 \nmanuscript.  636 \n 637 \nAcknowledgments 638 \nThis work was supported by grants to L.H.G. from the European Research Council (ERC 639 \nAgreement, TranscriptStress, 101076758), a Hallas -Møller Emerging Investigator Grant from 640 \nthe Novo Nordisk Foundation (NNF20OC0059959), and a Sapere Aude Research Leader 641 \nProgramme grant from the Independent Research Fund Denmark (0165 -00092B). Research at 642 \nthe Center for Gene Expression (CGEN) is funded by the Danish National Research Foundation 643 \n(DNRF166). Q.A.T. was supported by the Lundbeck Postdoctoral Fellowship Programme (LF 644 \nR380-2021-1284). N.N.M. was supported by the EMBO Postdoctoral Fellowship (ALTF 911-645 \n2020). Mass spectrometry -based proteomics analyses were performed by the Proteomics 646 \nResearch Infrastructure (PRI) at the University of Copenhagen (UCPH), supported by the Novo 647 \nNordisk Foundation (grant agreement number NNF19SA0059305). We thank members of the 648 \nCGEN/CPR/reNEW Sequencing Facility, University of Copenhagen, for expert technical 649 \nassistance, and Jesper Q. Svejstrup for feedback on the manuscript. 650 \n 651 \n 652 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 27 \nDeclaration of interest 653 \n 654 \nThe authors declare that they have no competing financial interests.  655 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n28 \n656 \n657 \n658 \n659 \n660 \n661 \n662 \n663 \n664 \n665 \n666 \n667 \n668 \n669 \n670 \n671 \n672 \n673 \n674 \n675 \n676 \n677 \n678 \n679 \n680 \n681 \nMethods \nResource availability \nPlasmids used in this study have been deposited in Addgene. Any additional information \nrequired to reanalyse the data reported in this paper is available from the lead contact \nupon request. \nExperimental Model \nFlp-InTM T-REx HEK293 cells (HEK293, R78007, ThermoFisher Scientific, human \nembryonic kidney epithelial, female origin) were cultured at 37 oC with 5% CO 2 in high \nglucose DMEM (Biowest, L0104) supplemented with 10% v/v FBS (Gibco, 10270106), 100 \nU/mL penicillin, 100 μg/mL streptomycin (Gibco, 10378016) 2 mM L-glutamine. Cells were \nroutinely passaged \n2-3 times a week. HEK293 CSB KO cells have previously been described 67. \nAll cell lines were\nconfirmed to be mycoplasma-free. Oxidative stress induction treatments were carried out for \n15 min (or 30 min for EU-labelling) with direct addition of freshly prepare 1000x concentrate \nto cell culture media, fresh H2O2 (maximum 3 month old kept at 4oC) to final concentration of \n1 mM H 2O2 (Sigma, H1009) or freshly resuspended Menadione sodium bisulfite (MND, \nSigma, M5750) in water to final concentration of 0.25 mM. After treatment the media was \ndiscarded, and cells were washed once with PBS pH = 7.4 (Gibco, 10010056) and replenished \nwith fresh 37oC cell culture media. Inhibitors treatments were carried out by direct addition to \nthe cell culture media with the following concentration 100 μM DRB (Sigma, D1916) or/and \n10 μM Olaparib (PARPi, MedchemExpress, Cat#HY-10162) or/and 10 μM TH5487 (OGG1i, \nMedchemExpress, Cat#HY-125276) and 10 μM PDD 00017273 (PARGi, MedchemExpress, \nCat#HY-108360). DRB inhibition was carried out 3.5 hours prior to other treatments. \nInhibition \n682 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 29 \nof PARPi, PARGi and OGG1i were preformed 1 hour prior to other treatments unless specified 683 \notherwise. Cells treated with inhibitors were subjected to oxidative stress by direct addition of 684 \noxidative reagents to cell culture media. PARPi, OGG1i and PARGi inhibitors were then 685 \nreplenished together with cell culture media during stress recovery. DRB was either replenished 686 \nor not depending on experiments. UV irradiation was performed with an exact dose of 20 J/m2 687 \nas in 10. Heat shock (HS) was performed by incubating cells at 43°C with pre-warmed media 688 \nfor 30 min or 1 hour. 689 \n 690 \nPlasmid construction and generation of stable cell lines 691 \nGeneration of stable cell lines 692 \nHEK293 Flp-In™ T-REx™ RPB3-3xFLAG cells (RPB3-FLAG) and U2OS Flp-In™ T-REx™ 693 \nGFP-NELFE were generated by stable integration of the pDEST/TO/FRT/RPB3 -3xFLAG or 694 \npFRT/TO/GFP-NELFE plasmids respectively. The human RPB3 (POLR2C) cDNA  was 695 \namplified from pIRES -puro-RPB3-HA plasmid (gifted from Jesper Svejstrup)  and NELFE 696 \ncDNA were PCR amplified with Q5® High -Fidelity DNA Polymerase (NEB) from HEK293 697 \nWT cells genomic DNA extracted with Quick -DNA™ Miniprep Plus Kit (Zymo Research). 698 \nRespective cDNA fragments were inserted through digestion and ligation into pENTR4 699 \ncontaining attB flanking sequences. RPB3 cDNA (in pENTR4 -RPB3) and NELFE cDNA (in 700 \npENTR4-NELFE) were tagged through insertion into respectively pDEST /TO/3xFLAG and 701 \npDEST/GFP/NELFE destination vectors using Gateway ™ LR Clonase II (Invitrogen, 702 \nThermoFisher Scientific), resulting with in -frame C -terminal fusion of RPB3 to 3xFLAG 703 \n(3xDYKDDDDK) tag and N -terminal fusion of NELFE with EGFP.  HEK293 Flp -In cells in 704 \nDMEM without antibiotic complementation were transfected for 48 hours with 900 ng insertion 705 \nplasmid pOG44 (ThermoFisher Scientific) and 100 ng donor plasmid pDEST/TO/RPB3 -706 \n3xFLAG or pDEST/GFP/NELFE using Lipofectamine ™ 3000 (Invitrogen, Therm oFisher 707 \nScientific; Cat#L3000015), following the manufacturer’s protocol. After 24 hours, the 708 \ntransfection cell culture media was replaced with fresh culture media for 24 hours, followed by 709 \none passage prior to selection with hygromycin (100 μg/mL), zeocin (100 μg/mL), and 710 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 30 \nblasticidin (15 μg/mL) for 10 days. Single colonies were manually picked and passaged 2–3 711 \ntimes prior to testing. Tagged cell lines were treated with doxycycline (100 ng/mL) for 24 hours 712 \nto induce RPB3-3xFLAG or GFP-NELFE confirmed by western blotting with respectively anti-713 \nFLAG (Sigma, Cat#F1804) and anti-GFP (Abcam, Cat#ab290) antibodies. 714 \n 715 \nGeneration of knock-out cell lines 716 \nHEK293 Flp -In™ T-REx™ XRCC1 knockout cells (XRCC1 KO) were generated by 717 \nCRISPR/Cas9-mediated genome editing. The pDG458 -XRCC1/sg1&2 plasmid was 718 \nconstructed by cloning two guide RNAs (gRNAs: 5′-TGCAGGACACGACATGGCGG-3′ and 719 \n5′-AGCCCACGACGTTGACATGC-3′) into BbsI-digested pDG458 containing Cas9 fused to 720 \nEGFP. This plasmid was transfected into HEK293 cells using Lipofectamine ™ 3000 for 48 721 \nhours according to manufacturer’s protocol. GFP-positive cells were then sorted using a FACS 722 \nsystem (FACSMelody ™, BD Biosciences) into two 96 -well plates. After 2 –3 passages, 723 \nindividual clones were screened by western blotting to confirm XRCC1 knockout. Two 724 \nvalidated knockout clones (KO#3 and KO#8) were used in subsequent experiments. HEK293 725 \nFlp-In™ T-REx™ XPC knockout cell s (XPC KO) line was generated by CRISPR/Cas9 -726 \nmediated genome editing. The gRNA sequence 5’ -CAACATGGCTCGGAAACGCG-3’ was 727 \nligated into the vector pSpCas9(BB) -2A-Puro (pX459) V2.0 (Addgene, #48138) to generate 728 \nthe pX459_XPC_gRNA1 plasmid. HEK293 were transfected with the plasmid 729 \npX459_XPC_gRNA1 using Lipofectamine TM 2000 for 48 hours following manufacturer 730 \ninstructions. Transfected cells were passaged and treated with puromycin (2 μg/mL) for 72 731 \nhours. Cells were then washed twice with PBS, detached with trypsin and quantified to prepare 732 \nsingle cell dilutions in a 96 well plate. Two weeks after, colonies originated from a single cell 733 \nwere evaluated for XPC knock out by western blot using the anti-XPC antibody (D1M5Y, Cell 734 \nSignalling, Cat#14768). Flp-In™ T-REx™ CSB knockout cell s (CSB KO) were previously 735 \ndescribed.10 736 \n 737 \nGeneration of NELFC degron cell line 738 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 31 \nHEK293 Flp-In™ T-REx FKBP12F36V-NELFCD cells (NELFCD-degron) were generated via 739 \nCRISPR double break following homology recombination knock -in of a C -terminal FKBP12 740 \ndegron tag, along with a cleavable puromycin resistance gene, into the endogenous HEK293 741 \nNELFCD locus with the donor plasmid pDONR -HA1/NELFCD/FKBP/PURO/HA2. To 742 \ngenerate the donor plasmid, NELFCD cDNA sequence and homology arms (HA1 and HA2; 743 \n>500 bp each) were amplified by PCR with  Q5® High -Fidelity DNA Polymerase from 744 \nHEK293 genomic DNA. The degron and puromycin resistance cassette were amplified from 745 \nthe pCRIS -PITChv2-Puro-dTAG (BRD4) plasmid (Addgene #91793). All fragments were 746 \nassembled into a donor plasmid (pBluescript II SK(+)) using Gibson assembly with the 747 \nNEBuilder® HiFi DNA Assembly Kit (NEB). To created double strand breaks and allow 748 \nhomology recombination targeted CRISPR components were delivered to cells, the pDG458 -749 \nNELFCD-sg1&2 plasmid was constructed by cloning two gRNAs (5′ -750 \nATTTGCAGTGAGCTTTAACG-3′ and 5′ -TGAAAGGGTTTTTCCACAAC-3′) into BbsI -751 \ndigested pDG458 (Addgene, #100900). HEK293 cells were then co-transfected with pDONR-752 \nHA1/NELFCD/FKBP/PURO/HA2 and pDG458 -NELFCD-sg1&2 using Lipofectamine TM 753 \n3000 with manufacturer protocols in antibiotic -free cell culture media. After 24 hours, cells 754 \nwere selected with puromycin (2 μg/mL) for 48 hours. For selection of positive clones, colonies 755 \nwere picked after selection passaged 2 -3 times and genomic DNA extracted with 756 \nQuickExtract™ DNA Extraction Solution (Lucigen , Cat# LGCQE09050). A PCR fragment 757 \noverlapping tagged region was amplified clones were selected based on fragment size shift and 758 \nfurther confirmed by western blotting with NELFCD -specific antibodies. To further validate 759 \nthe degron system, homozygous knock -in clones were treated with 250 nM dTAG -v1 (Tocris 760 \nBioscience, Cat#6914) for 1 hour to induce in vivo  NELFCD degradation. Depletion of 761 \nNELFCD protein and reduction of chromatin-bound NELFA protein levels were confirmed by 762 \nwestern blotting. 763 \n 764 \nEU-labelling assay 765 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 32 \nApproximately 7,000 HEK293 cells were seeded in 96-well Greiner CELLSTAR® 96 well plate 766 \n(ThermoFischer Scientific, Cat#M0562) coated for 15 min at room temperature  with poly-D-767 \nLysine (Gibco, Cat#A3890401) and washed twice with PBS. For treated samples, cell culture 768 \nmedia was replaced by media complemented with indicated concentration of H2O2 or MND for 769 \n30 min and washed with PBS and replenished with new 37oC cell culture media. Nascent RNA 770 \nwas pulse-labelled for 30 min with addition of cell culture media complemented with 5 -771 \nEthynyluridine ( EU, Jena Bioscience, Cat#CLK -N002, final concentration 750 μM). For 772 \ntreated samples  (no recovery)  both oxidative reagents and EU were added simultaneously. 773 \nLabelled cells were immediately fixed with 3.7  % formaldehyde diluted in PBS for 45 min in 774 \nthe dark. The cells were then washed three times in PBS and permeabilized with 0.5  % Triton 775 \nX-100 in PBS for 30 min at room temperature in the dark. Nascent RNA was then visualized 776 \nby click-it chemistry, the EU-incorporated RNA was labelled in the dark for 2 hours with a mix 777 \nof 5 μM Alexa Fluor TM 488 Azid (ThermoFischer Scientific , Cat# A10266), 4 mM copper 778 \nsulfate (Sigma-Aldrich) and 100 mM ascorbic acid (Sigma -Aldrich). Cells were then washed 779 \ntwice with PBS and incubated for 30 min at room temperature in the dark with 2 μg/mL DAPI 780 \n(Sigma-Aldrich, Cat#D9742) diluted in PBS. Cells were washed twice with PBS and kept in 781 \nthe dark at 4oC in PBS for up to 48 hours. A total of 25 images per well were acquired with the 782 \nScanR acquisition system (Olympus) controlling a motorized Olympus IX -81 wide -field 783 \nmicroscope and analysed and quantified with the ScanR Analysis software. 784 \n 785 \nTotal RNA extraction 786 \nTotal RNA was extracted by adding TRIzol ™ Reagent ( ThermoFisher Scientific , 787 \nCat#15596026) directly to the cell monolayer after cell culture media removal. Chloroform was 788 \nadded to the cell/TRIzol mixture at a 1:5 volume ratio, followed by thorough mixing for 30 789 \nseconds and centrifugation at 12,000 g  for 15 min at 4 °C. The upper aqueous phase was 790 \ncollected and further purified by adding 1 volume of chloroform/isoamyl alcohol (24:1; Sigma-791 \nAldrich, Cat#C0549), followed by vortexing and centrifugation at 12,000 × g for 5 min at 4 °C. 792 \nThe resulting upper phase, containing total RNA, was precipitated by adding one volume of 793 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 33 \nisopropanol (Sigma -Aldrich, Cat#I9516) and 30 ng/mL GlycoBlue ™ (Invitrogen, 794 \nThermoFisher Scientific, Cat#AM9515) and incubated at room temperature for 20 min. The 795 \nRNA was pelleted by centrifugation at 20,000 g for 30 min and washed once with ice-cold 85% 796 \nethanol. The final RNA pellet was resuspended in RNase-free water. 797 \n 798 \nNascent RNA dot blot 799 \nDot blots were used to detect global levels of nascent transcription based on 4-thiorudine (4sU, 800 \nGlentham Life Sciences, Cat#GN6085) RNA  incorporation. Dot blot  were performed as 801 \ndescribed in 38 with minor changes. Approximately 500,000 cells were seeded in 6-well plates. 802 \nThe next day, 4sU (final concentration 1mM) was added directly to the tissue culture for 15 803 \nmin coordinated with H2O2 treatment and recovery time points, labelling was then arrested with 804 \naddition of 0.5 mL TRIzol. Total RNA was extracted and 3 μg of 4sU labelled total RNA was 805 \nbiotinylated for 2 hours in the dark with 50 μL of 0.1 mg/ml EZ -linkTM HPDP-Biotin 806 \n(ThermoFischer Scientific, Cat#21341 in DMF) with 3 μL of biotin buffer (833 mM Tris -HCl 807 \npH 7.4, and 83.3 mM EDTA) for a final reaction volume of 300 μL. Unreacted biotin-linker 808 \nwas removed from RNA samples  by addition of 1.1 volumes of ROTI ®Aqua-P/C/I (Roth, 809 \nCat#985.1) vortexed, precipitated with 1.1 isopropanol followed by pellet centrifugation, 810 \nethanol washes and resuspension in RNAse-free H2O. Purified RNA sample were resuspended 811 \nin 10 μL and applied to a Hybond -N+ membrane  (Cytiva, Cat#RPN203B ) in a dot blot  812 \napparatus (BioRad). The immobilized RNA was UV-crosslinked to the membrane with UV-C 813 \n0.24 J/cm2 and the membrane incubated in blocking solution (PBS pH = 7.4; 10% SDS; 1 mM 814 \nEDTA) for 20 min at room temperature followed by at 15 min incubation with a 1:50,000 815 \ndilution of 1  mg/mL streptavidin -horseradish peroxidase (HRP) ( ThermoFisher Scientific 816 \nCat#N100) in blocking solution. The membrane was then washed twice in blocking solution 817 \nbuffer for 10 min, followed by two washes in dot blot wash buffer I (PBS pH = 7.4; 1% SDS) 818 \nfor 10 min each and two washes in dot blot wash buffer II (PBS pH = 7.4 ; 0.1% SDS) for 10 819 \nmin each. The biotin -bound HRP was visualized with 2 min incubation of 1:4 diluted ECL 820 \ndetection reagent (BioRad) on an ChemiDoc Imaging System (BioRad). As a loading control, 821 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 34 \nthe membrane was stained with methylene blue solution (0.5  M sodium acetate; 0.5  % 822 \nmethylene blue) for 10 min, then de-stained by several washes in water. 823 \n 824 \nTTchem-seq 825 \nTTchem-seq was performed as described previously with minor modifications 38. In brief, 10 cm 826 \ndishes with 80% confluent cells  RNA were labelled in vivo for 15 min with 4sU by adding it 827 \ndirectly to the tissue culture media to a final concentration of 1 mM. Labelling was terminated 828 \nby addition of TRIzol and total RNA was extracted by TRIzol/chloroform extraction followed 829 \nby isopropanol precipitation. Per samples 80 -100 μg of total RNA resuspended in 100 μL 830 \nRNAse-free H 2O was complemented with 1  % ( relative to total RNA concentration) of S. 831 \ncerevisiae 4tU-labelled RNA spike-in. Total RNA was fragmented by adding 20 μL of freshly 832 \nprepared 1 M NaOH per sample, thoroughly mixed and incubated for 40 min on ice to < 400 nt 833 \nfragments. RNA fragmentation was terminated by addition of 80 μL 1 M Tris -HCl pH = 6.8 834 \nand samples were immediately purified with 1 volume of ROTI ®Aqua-P/C/I (ROTH, Cat#  835 \nX985.1) extraction and precipitated with isopropanol. Resuspended and purified 4sU labelled 836 \nRNA in RNAse -free water was biotinylated in 10 mM Tris -HCl pH = 7.4; 1 mM EDTA 837 \ncomplemented with 0.5 mg/ μL MTSEA biotin-XX linker (Biotum, Cat# BT90066) for 30 min 838 \nin the dark at room temperature. Biotinylated RNA was immediately purified by ROTI®Aqua-839 \nP/C/I extraction and isopropanol precipitation and resuspended in 50 μL RNAse-free water. To 840 \nenrich for 4sU labelled RNA, the purified RNA was denatured at 65°C for 10 min, with 5 min 841 \non ice was used, and isolated with μMACS Streptavidin Kit (Miltenyi, Cat#130 -074-101). A 842 \ntotal of 100uL μMACS Streptavidin MicroBeads were added to denatured 4sU total RNA and 843 \nincubated for 15 min at room temperature with agitation. Beads bound to 4sU biotinylated RNA 844 \nwere applied to magnetic μColumns and transferred to the magnetic field of the μMACS 845 \nmagnetic separator (Miltenyi). The columns were washed t wice with 1 mL 55oC pre-warmed 846 \npull-down wash buffer (100 mM Tris -HCl pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1  % (v/v) 847 \nTween 20) and eluted with two consecutive washes with freshly prepared 100  mM DTT. 848 \nBiotinylated RNA was further cleaned up from DTT elution by ROTI®Aqua-P/C/I extraction 849 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 35 \nand isopropanol precipitation  and resuspended in RNAse -free H 2O. Concentration of 4sU 850 \nenriched RNA was measured by Qubit (ThermoFischer Scientific , Cat# Q32852) using the 851 \nRNA HS assay kit and size of fragmented RNA assessed by TapeStation using RNA 852 \nScreenTape Assay for TapeStation Systems (Agilent, Cat# 5067-5576). 30 ng of purified 4sU 853 \nbiotinylated RNA was used to prepare libraries with NEBNext® Ultra ™ II RNA library prep 854 \nkit (NEB, Cat#E7760) according to manufacturer’s protocol (protocol 5 for fragmented RNA) 855 \nwith ligation of two barcodes of 8  nt and 11 nt UMIs (NEB, Cat#E7416). Libraries were 856 \namplified using 8 cycles of PCR and sequenced as single -end 68 bp reads on a NextSeq 2000 857 \nplatform (Illumina).  858 \n 859 \nCell growth assays 860 \nApproximately 5,000 HEK293 cells per well were seeded in triplicates in  96-well plates, pre-861 \ncoated with poly-D-Lysine (Gibco) for 15 min and washed twice with PBS. The following day 862 \ncell treatments were performed in 96 -wells by addition of cell culture media complemented 863 \nwith different concentration of H 2O2 or MND for 15 min. The complemented media was 864 \nremoved, and cells were washed once with PBS and fresh 37 oC cell culture media was 865 \nreplenishment. Growth was monitored using an IncuCyte S3 Live -Cell Analysis System 866 \n(Sartorius). Images of live cells were captured every 2 hours for up to 6 days. Images were 867 \nanalysed in the IncuCuyte S3 image analysis software, with an output of cell confluency (%). 868 \n 869 \nCell fractionation and RNAPII immunoprecipitation 870 \nCell pellets from one 15 cm plate were resuspended in 1 mL volume HE buffer (10 mM HEPES-871 \nNaOH pH=7.5; 10 mM KCl, protease and phosphatase inhibitors: Vanadate, Sodium fluoride, 872 \nand ß-glycerolphosphate) and incubated 15 min on ice. Nuclei were pelleted 15 min; 1,000 g  873 \nand the supernatant taken as the cytoplamic fraction. The nuclear pellet was resuspended in 500 874 \nml NE buffer (20 mM HEPES -NaOH pH = 7.9; 150 mM NaCl; 0.05% NP40; 10% glycerol; 875 \nprotease and phosphatase inhibitors fresh). Chromatin were pelleted 15 min; 20,000 g and the 876 \nsupernatant taken as the nucleoplasmic fraction. Cytoplasmic and nucleoplasmic fractions were 877 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 36 \npooled as soluble fraction. Chromatin pellets were digested in 500 m L of CD Buffer (20 mM 878 \nHEPES-NaOH pH=7.9; 150 mM NaCl; 1.5 mM MgCl 2; 0.05% NP40; 10% glycerol; protease 879 \nand phosphatase inhibitors fresh; 250 U/ml DENARASE® (c-Lecta, Cat#20804) and incubated 880 \non a rotating wheel for 1 hour at 4oC to digest nucleic acids and solubilize chromatin and release 881 \nchromatin-bound proteins. Digested chromatins were pelleted 15 min at 20,000 g and the 882 \nsupernatant was taken as the enriched chromatin fraction. For immunoprecipitation, 883 \nDynabeadsTM Protein G Magnetic Beads (Invitrogen, ThermoFischer Scientific, Cat#10004D) 884 \nwere washed and resuspended in 2 beads volumes of PBS + 0.02% Tween 20. The resuspended 885 \nbeads were conjugated for 1 hour at room temperature with 1 mg 4H8 or 3E10 as indicated (for 886 \ncapture of phosphorylated RNAPII complexes) or with anti-FLAG (Sigma, #1806, for capture 887 \nof total RNAPII complexes in a RPB3 -FLAG expressing cell line ). Non-antibody conjugated 888 \nbeads were used for pull -out as negative control for mass spectrometry. Following  antibody 889 \nincubation, unconjugated antibodies were removed by from beads by two washes with PBS + 890 \n0.02 % (v/v) Tween20. Protein concentrations of chromatin enriched fractions were measured 891 \nwith Protein Assay Dye Reagent (BioRad, Cat#5000001) through absorbance measured at 495 892 \nnm and concentration was adjusted to the sample with the lowest concentration by addition of 893 \nCD buffer (constituting the ‘input’ sample for western blotting). Concentration -adjusted 894 \nchromatin fractions were added to antibody-conjugated beads  (or unconjugated beads for 895 \nnegative controls) and incubated for 3 hours at 4 oC with agitation. Samples were placed on a 896 \nmagnetic rack and the supernatant was removed and kept  (‘unbound’ lysate for western 897 \nblotting). Proteins bound to magnetic beads were then washed three times with IP wash buffer 898 \n(10 mM Tris -HCl pH=7.5; 150 mM NaCl; 0.05% NP40; 0.5 mM EDTA; protease and 899 \nphosphatase inhibitors fresh). Beads bound proteins were either extracted by boiling with 900 \n2xSDS sample buffer ( 10 mM Tris -HCl; pH = 6.8, 4% SDS, 0.2% bromophenol blue, 20% 901 \nGlycerol, 200 mM DTT) for western blotting (‘IP’ sample) or for mass spectrometry analysis, 902 \nthe beads were  washed two additional times with TBS and stored dry at  -20oC until further 903 \nprocessing for proteomics. 904 \n 905 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 37 \nMass spectrometry 906 \nAll samples for proteomic analysis were carried out in triplicate and samples were prepared in 907 \nparallel from the point of cell seeding. TBS washed frozen beads were thawed and incubated 908 \nfor 30 min with elution buffer 1 (50 mM Tris -HCl pH = 7.5; 2 M Urea; 2 mM DTT; 20 μg/ml 909 \ntrypsin) followed by a second elution for 5 min with elution buffer 2 (50 mM Tris -HCl pH = 910 \n7.5; 2 M Urea; 10 mM Chloroacetamide). Both eluates were combined and further incubated 911 \nat room temperature over-night. Tryptic peptide mixtures were acidified to 1% TFA and loaded 912 \non Evotips (Evosep). Peptides were separated on 15 cm, 150 μM ID columns packed with C18 913 \nbeads (1.9 μM) (Pepsep) on an Evosep ONE HPLC applying the ‘30 samples per day’ method 914 \nand injected via a CaptiveSpray source and 10 μm emitter into a timsTOF pro mass 915 \nspectrometer (Bruker) operated in PASEF mode.  916 \n 917 \nWestern blotting 918 \nFor whole cell extracts, cell pellets were collected, resuspended in whole cell extraction buffer 919 \n(20 mM Tris -HCl pH = 7.5; 300 mM NaCl; 2.5 mM MgCl2; 1% NP40; 10% glycerol) with 920 \nfreshly added DENARASE® (c-Lecta, Cat#20804), fresh protease and phosphatase inhibitors, 921 \nand then incubated rotating at 4°C for 1 hour followed by centrifugation at 10,000 g for 2 min 922 \nat 4°C. Supernatant was transferred to a new tube and protein concentration measured using 923 \nBio-Rad protein assay reagent. For whole cell extracts , chromatin enriched fractions and IP 924 \nfractions, protein concentrations were adjusted to the sample with the lowest concentration with 925 \nthe respective appropriate extraction buffer. 10 μL protein samples was complemented with 10 926 \nμL 2x SDS sample buffer  and boiled at 98°C for 5 min. 20 μL  protein sample were separated 927 \nby SDS-PAGE gel electrophoresis (6%, 8%, 10% or 15% acrylamide were used) in running 928 \nbuffer (25 mM Tris pH = 8.3, 192 mM glycine, 0.1% SDS,) and transferred to nitrocellulose 929 \nmembranes by wet transfer in transfer buffer (0.025 M Tris, 0.192 M glycine, 10% (v/v) 930 \nethanol). Membranes were stained with Ponceau S solution for 10 min and washed with dH2O, 931 \nand a picture was acquired. Membranes were then blocked in blocking buffer (5% (w/v) milk 932 \nin TBS-T) and incubated with primary antibody (in 5% (w/v) milk in TBS-T) overnight at 4°C. 933 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 38 \nMembranes were then washed with TBS -T, and incubated with HRP -conjugated secondary 934 \nantibodies in blocking buffer for 1 hour at room temperature and then washed several times in 935 \nTBS-T. HRP signal detection was obtained using 1:1 ratio of each Clarity ECL Western 936 \nBlotting Substrates (BioRad, Cat#1705061) applied for 2min onto the membrane and 937 \nvisualization by ChemiDoc Imaging System (BioRad).  938 \n 939 \nELCAP-seq 940 \nELCAP-seq was carried out similarly to previously des cribed 39. RPB3 -FLAG cells were 941 \nseeded in 4x15 cm dished per samples; RPB3-FLAG was induced by adding a final 942 \nconcentration of 100 ng/mL doxycycline for 16 hours directly to cell culture media. Induced 943 \ncells were treated with 1 mM H2O2 for 15 min or let recover after a PBS wash and replenishment 944 \nof warm cell culture media for variable recovery times. Immediately cells were collected, 945 \ncentrifuged for 2 min at 1,000 g and the pellets frozen in liquid nitrogen. Frozen cells were then 946 \nimmediately used for cell fractionation as described above. A volume of 25 μL  DynabeadsTM 947 \nProtein G Magnetic Beads per sample were washed and conjugated to 1 mg anti-FLAG (Sigma, 948 \n#1806) as previously described in immunoprecipitation section. Protein concentrations from 949 \nchromatin enriched fraction were measured with Protein Assay Dye Reagent (BioRad) and 950 \nadjusted to the sample with the lowest concentration with CD buffer. Normalised chromatin 951 \nenriched fractions were then added to FLAG-conjugated magnetic beads for 3 hours at 4oC with 952 \nagitation. The beads were washed 4 times with ELCAP wash buffer (10 mM Tris -HCl pH = 953 \n7.5; 250 mM NaCl; 0.5% (v/v) NP -40; 0.5 mM EDTA; fresh protease inhibitor; protease 954 \nInhibitors cocktail and fresh phosphatase inhibitors). Short RNA  fragments protected by  955 \nRNAPII were extracted with the Quick -RNA MicroPrep kit (Zymo, Cat#R1050) according to 956 \nthe manufacture’s instruction  for short RNA extraction (17 -200 nt). Briefly, RNPAII bound 957 \nbeads were incubated for 5 min at RT in extraction buffer (per  100 μL sample; 100 μL of 958 \nsupplied Zymo RNA lysis Buffer, 100 μL of IP wash buffer,100 μL ethanol). RNA was isolated 959 \nusing kit supplied Zymo-Spin™ IC columns following the manufacturer’s instructions. The 960 \nsize distribution of extracted  RNA was assessed with  the Bioanalyzer Small RNA Analysis 961 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 39 \nsystem (Agilent). Library were prepared using 30 ng of short RNAs with NEBNext® Small 962 \nRNA library preparation kit (NEB, Cat# E7300) according to the manufacturer’s protocol with 963 \nthe only modification being inclusion of a UMI containing adapter ; the i nitial ligation was 964 \ncarried with the custom ligation adapter ‘5' -App-965 \nNNNNNNAGATCGGAAGAGCACACGTCT-3' (IDT) with the same ligation conditions as 966 \nmanufacturer’s protocol. Libraries were amplified with 8 cycles of PCR and the whole library 967 \nwas separated by size with Novex™ TBE Gels, 6% (Invitrogen, ThermoFischer Scientific, Cat# 968 \nEC6265BOX), stained with SYBRTMgold (Invitrogen, ThermoFischer Scientific, Cat# S11494) 969 \nand fragment size ranging from 147  nt to 190  nt were excised from the gel and purified 970 \naccording to NEBNext® Small RNA library preparation kit protocol. Size selected libraries 971 \nwere then sequenced as single-end run (68 bp reads) on a NextSeq2000 platform (Illumina). 972 \n 973 \nUbiquitinated proteins pull-down 974 \nDSK2 beads were prepared as previously described 40 with minor changes. In brief E. coli 975 \nexpressing DSK2 were diluted in ice cold dilution buffer (1x PBS, 0.5% Triton X-100, protease 976 \ninhibitors) and sonicated (15 s ON, 30 s OFF, total 10 min ON time). HEK293 cells were either 977 \ntreated with UV (20 J/m2) and let recover for 45 min after cell culture media replenishment or 978 \nwith 1 mM H 2O2 for 15 min and recovery time as indicated. Cell pellets were resuspended in 979 \nTENT buffer (50 mM Tris-HCl pH = 7.4; 2 mM EDTA; 150 NaCl; 1 % Triton X-100; 2,5 µM 980 \nMgCl; protease and phosphatase inhibitors added fresh and 250  U/ml DENARASE® ) and 981 \nincubated 1 hour at 4 oC. Debris and non-solubilized proteins were removed by centrifugation 982 \nat 14,000 g for 10 min. Protein concentration of the resulting supernatant was determined with 983 \nProtein Assay Dye Reagent (Biorad). Lysates protein concentration was equalized before 984 \nadding 25 µL DSK2 beads and incubated overnight at 4oC. DSK2 beads enriched for ubiquitin 985 \nbound proteins were spun down at 700 g for 5 min and washed once in TENT buffer and once 986 \nwith PBS (pH = 7.4). To release ubiquitinated proteins from the beads, samples were heated at 987 \n95oC for 5 min in 2x SDS sample buffer . The samples were separated from the beads by 988 \ncentrifugation and 1% input and 20 % sample were used for western blotting. 989 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 40 \n 990 \nChromatin immunoprecipitation and sequencing (ChIP-seq) 991 \nFor each sample, 80 % confluent HEK293 cells in a 15 cm plate were crosslinked with 1 % 992 \nformaldehyde for 15 min and stopped with 125 mM glycine for 5 min. Fixed cells were then 993 \nhomogenized in cellular lysis buffer (5 mM Pipes pH = 8; 85 mM KCl; 0.5 % NP‐40). Nuclei 994 \nwere then resuspended in nuclear lysis buffer (50 mM Tris pH = 8.1; 10 mM EDTA; 1 % SDS) 995 \nand sonicated to obtain DNA fragments between 50 bp and 500 bp (5 cycles; 30sec ON, 30sec 996 \nOFF, with Bioruptor pico). Samples were diluted 10 times in dilution buffer (0.01 % SDS; 1.1 997 \n% Triton X‐100; 1.2 mM EDTA; 16.7 mM Tris pH = 8; 167 mM NaCl). Samples were pre -998 \ncleared with 25 µL proteins A/G beads (ThermoFisher Scientific, Cat# 20422 ) and blocked 999 \nwith 500 µg BSA for  each 200 µg of chromatin for 2 hours at 4°C. A volume of 100 µL pre -1000 \ncleared chromatin was keep at -20°C for inputs and another volume of chromatin was then 1001 \nincubated overnight with either NELFE or total RPB1 (D8L4Y) antibodies at 4°C. 1002 \nImmunoprecipitation was performed using 35 µL proteins A/G beads (blocked with 500 µg 1003 \nBSA) for 2 hours at 4°C. Beads were then washed once with dialysis buffer (2 mM EDTA; 50 1004 \nmM Tris-HCl pH=8.1; 0.2% Sarkosyl), once wash buffer (100 mM Tris pH = 8.8; 500 mM 1005 \nLiCl; 1% NP‐40; 1% NaDoc) and once with TE (10 mM  Tris pH = 8; 0.5 mM EDTA). 1006 \nImmunoprecipitated complexes and inputs were resuspended in TE and incubated with 1 µL of 1007 \nRNase A (10 mg/mL , ThermoFisher Scientific, Cat# EN0531) for 30 min at 37°C. Reverse 1008 \ncrosslinking was performed by incubating samples overnight at 70°C with 4 µL SDS (10%) 1009 \nand incubation with ProteinaseK (10 mg/mL, ThermoFisher Scientifc Cat#AM2548) for 1 hour 1010 \n30 min at 45°C. DNA was purified by phenol/chlorophorm extraction followed by ethanol 1011 \nprecipitation. Libraries were prepared with NEBNext® Ultra™ II DNA library prep kit (NEB, 1012 \nCat#E7604) with 12 nt UMIs adaptors (NEB, Cat#E7395) according to manufacturer’s protocol 1013 \nand sequenced on a NextSeq2000 platform (Illumina). 1014 \n 1015 \nIn vitro transcription with pig thymus RNAPII 1016 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 41 \nRNAPII was purified from pig thymus as previously described 68. The elution of the 8WG16 1017 \nconjugated beads was dialyzed in dialyze buffer (20 mM HEPES-NaOH pH = 8; 150 mM NaCl; 1018 \n10 % Glycerol; 2 mM 𝛽-mercaptoethanol) and concentrated with Amicon® Ultra 4 centrifugal 1019 \nfilter (50 kDa) (Millipore, Cat#UF8010). The elongation complex was assembled as previously 1020 \ndescribed with minor modification 69. Briefly, 5 pmol of pre -annealed RNA:DNA (template 1021 \nstrand) hybrid was mixed with an equimolar amount of pure RNAPII and incubated 20 min at 1022 \n30oC, followed by the addition of 10 pmol 5’ biotin -labelled non-template strand DNA for an 1023 \nadditional 10 min. The assembled elongation complex was diluted with transcription buffer (20 1024 \nmM Tris -HCl pH= 7.5; 100 mM NaCl; 8 mM MgCl2; 10 μM ZnCl2; 10% glycerol). The 1025 \ntranscription reaction was started by addition of 1 mM rNTPs for 2 min at 25 oC and stopped 1026 \nwith 20 mM EDTA, 0.5 μL Proteinase K and 45% formamide. For treatment, H2O2 was added 1027 \nbefore the rNTPs at the indicated concentrations. Samples were incubated for 30 min at 50 oC 1028 \nfollowed by 10 min at 70 oC. RNA products were resolved in 8 M Urea, 16% polyacrylamide, 1029 \n1x TBE gel. Gels were scanned in a Typhoon scanner (Cytiva) to detect Cy2 fluorescence. 1030 \n 1031 \nImmunofluorescence microscopy 1032 \nGFP-tagged NELFE U2OS cells were seeded (with 100 ng/mL doxycycline) at 60 -70% 1033 \nconfluency onto coverslips coated with Poly-D-Lysine (Gibco) in 12-well plates. The next day 1034 \ncells were treated for 15 min or 30 min with 1 mM H 2O2 and for 30 min or 1 hour heat  shock 1035 \nat 43oC. Cells were then fixed with 4% (v/v) formaldehyde for 15 min at room temperature, 1036 \nwashed once with PBS, and mounted onto slides using VECTASHIELD® Antifade Mounting 1037 \nMedium with DAPI (VWR, Cat#VECTH-1200). GFP and DAPI fluorescence were visualised 1038 \nwith an inverted confocal microscope (Zeiss LSM 900) 1039 \n 1040 \nImage analysis 1041 \nFor EU-labelling experiments, scanR software was used to quantify each isolated nuclei green 1042 \nsignal relative to DAPI signal. For picture plotting, raw tiff images were upload to ImageJ’s 1043 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 42 \nFiji, untreated samples (HEK293 or XRCC1 KO cells) were corrected for brightness and 1044 \ncontrast; the same correction was then applied to all other images of the same cell line.  1045 \n 1046 \nTTchem-seq analysis 1047 \nRaw fastq files for Read 1 were labelled with appropriate UMI reads using UMItools 70(v1.1.4) 1048 \nextract function followed by adapter trimming with cutadapt  (v4.5) within trim_galore 1049 \n(v0.6.10) and read length filtering > 19nt. For the purposes of visualization, biological 1050 \nreplicates UMI and adapter trimmed reads fastq files were merged and processed further 1051 \nsimilarly to individual replicates. In the case of XRCC1 KO, two individual clones (KO#3 and 1052 \nKO#8) were considered as biological duplicates. Merged and individual samples were mapped 1053 \nwith STAR 71 (v2.7.11b) against a merged human -yeast genome assemblies ( Homo sapiens  1054 \nGRCh38 and Saccharomyces cerevisiae sacCer, Ensembl release 108) with options as default. 1055 \nGenome alignments BAM files were filtered for multimapped reads with samtools (v1.21) view 1056 \nfunction (-q 10). UMI duplicates were then removed with UMItools dedup function. A yeast 1057 \ngene-level count matrix was generated with Subread (v2.0.6) featureCounts 72 and used to 1058 \ncalculate yeast size -factors with DEseq2 73 (v1.42.0) “estimateSizeFactors” function. Strand 1059 \nspecific and spike -in normalised BigWig files were generated from strand -specific bam files 1060 \nusing Deeptools74 (v3.5.5) bamCoverage function (--scaleFactor = 1/ yeast size-factor) with bin 1061 \nsize of 25 nt ( -bs 25). TTchem-seq scaled plots (metagene profiles or heatmaps) were created 1062 \nfrom spike-in normalised BigWig files with DeepTools computeMatrix function (scale-regions, 1063 \nbetween –2 kb TSS to TES + 2 kb, bin size 50bp) using gene definitions from Ensembl (release 1064 \n108). All gene features were selected for protein coding genes non-overlapping (extended to 5 1065 \nkb upstream the TSS and 5 kb downstream of the TES) other coding genes (n = 11,593). ‘Long 1066 \nGenes’ derived from the later and were filtered with length > 90 kb (n  = 2,217). Unscaled 1067 \n(reference-point) mode metagene profiles were centred on TSS with various distance around 1068 \nthe TSS. TT chem-seq signal relative to u ntreated control ratio BigWig files were created using  1069 \nthe DeepTools bigwigCompare function; the genome was partitioned to 100 bp bins for scaled 1070 \nmetagene profiles and 50 bp bins for unscaled metagene profiles. Heatmaps were sorted by 1071 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 43 \ngene length (decreasing or increasing). Quantitative differential expression analysis of TTchem-1072 \nseq data for Supplementary Fig. 2d was done through DESeq2 with replacement of human 1073 \nestimateSizeFactor by yeast spike-in size-factors. For Supplementary Fig. 5b BigWig files were 1074 \nconverted to bedGraph, forward and reverse strand were then merged into one bedGraph file. 1075 \nIn R (v4.3.2), with a custom script, coverage values were extracted from each feature with 1076 \nregards to strand and normalised to region size. Genomic spike-in normalised track screenshots 1077 \nwere exported from IGB as vector images. 1078 \n 1079 \nELCAP-seq analysis 1080 \nRaw fastq files were trimmed with cutadapt (v4.5) within trim_galore (v0.6.10) and minimum 1081 \nread length filtering > 19nt and maximum read length < 47 nt. For purposes of visualization, 1082 \nbiological replicates adapter trimmed and size filtered reads fastq files were merged and 1083 \nprocessed further similarly to individual replicates. Resulting reads were mapped with STAR 1084 \nwith ( –-alignEndsType Extend5pOfRead1) against the human genome assembly (GRCh38, 1085 \nEnsembl release 108). Genome alignment BAM files were filtered for multimapped reads and 1086 \nseparated by strand with samtools view function (-q 10, -F 16 or -f 16). Single nucleotide strand 1087 \nspecific and RPKM (read per million kilobase) normalised BigWig files were generated using 1088 \nDeeptools’ bamCoverage function for each stranded BAM files in single nucleotide resolution 1089 \nmode with the following options “ -bs 1 --Offset -1 --normalizeUsing RPKM”. ‘Pause sites’ 1090 \nwere obtained by calculating the highest peak position within pausing region ( -20 nt , TSS, 1091 \n+120 nt) in the untreated sample with CAGEfightR75 . 1092 \nELCAP-seq centred metagene plots (profiles or heatmaps) were created using deepTools 1093 \ncomputeMatrix in reference -point mode ( centred on TSS or the ‘pause sites’) over the same 1094 \nnon-overlapping protein coding gene set as for TT chem-seq plots. ELCAP-seq signal relative to 1095 \nuntreated control ratio BigWig files were created using the deepTools’ bigwigCompare 1096 \nfunction in single nucleotide mode ( -bs 1). Heatmaps were sorted by gene length (decreasing 1097 \nor increasing). For Supplementary Fig. 5c ratio between TT chem-seq and ELCAP -seq BigWig 1098 \nfiles for each time points were created using deepTools bigwigCompare function and scaled to 1099 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 44 \na normali sing ratio calculated for library size dispersion between the two datasets. For 1100 \nSupplematery Fig. 5d ELCAP-seq BigWig files were converted to bedGraph, forward and 1101 \nreverse strand were then merged into one bedGraph file. In R, with a custom script, the pausing 1102 \nindex (PI) was calculated as ratio of coverage values from TSS (TSS + 200 bp) over coverage 1103 \nfor gene body region (TSS + 200 bp to TES – 200 bp) relative to size. The recurrence of pausing 1104 \nindex was then plotted as distribution. 1105 \n 1106 \nMass spectrometry analysis 1107 \nRaw mass spectrometry data were analysed with MaxQuant (v1.6.15.0). Peak lists were 1108 \nsearched against the human Uniprot  FASTA database combined with 262 common 1109 \ncontaminants by the integrated Andromeda search engine. False discovery rate was set to 1  % 1110 \nfor both peptides (minimum length of 7 amino acids) and proteins. “Match between runs” 1111 \n(MBR) was enabled with a Match time window of 0.7, and a Match ion mobility window of 1112 \n0.05 min. Relative protein amounts were determined by the MaxLFQ algorithm with a 1113 \nminimum ratio count of two. Statistical analysis of LFQ derived protein expression data was 1114 \nperformed using the automated analysis pipeline of the Clinical Knowledge Graph. 93 Protein 1115 \nentries referring to potential contaminants, proteins identified by matches to the decoy reverse 1116 \ndatabase, and proteins identified only by modified sites, were removed. LFQ intensity values 1117 \nwere normalised by log2 transformation and proteins with less than 70 % of valid values in at 1118 \nleast one group were filtered out. The remaining missing values were imputed using the 1119 \nMinProb approach (random draws from a Gaussian distribution; width = 0.2 and downshift = 1120 \n1.8) or with Mixed imputation using KNN. 1121 \n 1122 \nChIP-seq analysis 1123 \nNELFE, total RPB1 and input raw fastq files were trimmed with with trim_galore (paired-end 1124 \nmode) with minimum read length filtering > 19 nt. Filtered and trimmed paired end reads were 1125 \nmapped with STAR against human assembly GRCh38 (Ensembl release 108). Genome 1126 \nalignment BAM files were filtered for multi-mapped reads with samtools view function (-q 10). 1127 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 45 \nBam files for NELFE or total RPB1 samples were normalised to input with MACS76 (v2.2.9.1). 1128 \nResulting bedGraph files were used to compute heatmaps. In Fig. 4e input normalised NELFE 1129 \nChIP-seq signal levels were used to sort (in decreasing order) both bottom panels of ELCAP -1130 \nseq signals. In Fig. 4f ChIP-seq ratio (sample relative to untreated control) BigWig files were 1131 \ncreated using the deepTools bigwigCompare function, the genome was partitioned to 50 bp 1132 \nbins. NELFE ChIP-seq ratio BigWig files were then normalised to total RPB1 ChiP-seq levels 1133 \nwith bigwigCompare function and plotted relative to NELFE ChIP-seq decreasing signal. 1134 \n 1135 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n 46 \nSupplemental item titles 1136 \n 1137 \nTable S1: Mass spectrometry of RNAPII immunoprecipitation experiments  1138 \nTable S2: List of oligonucleotides 1139 \n 1140 \n 1141 \n.CC-BY 4.0 International licenseperpetuity. 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Genome Biol 9, R137. 10.1186/gb-2008-9-9-r137. 1375 \n 1376 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\nFig. 1. Oxidative stress represses global nascent transcription of RNAPII without release from \nchromatin. a Schematic of oxidative stress inductions using either H2O2 or Menadione (MND). b \nNascent transcription levels measured by nascent RNA metabolic pulse labelling (15 min) with 4 -\nthiouridine (4sU). Oxidative stress was induced for 15 min in HEK293 cells with 1 mM H 2O2 or 0.25 \nmM MND, and cells were allowed to recovery for indicated times.  Staining with methylene blue as a \nloading control. c-d Nascent transcription levels quantified by 5 -Ethynyluridine (EU) labelling and \nsingle cell quantification by immunofluorescence microscopy (EU-assay). Per nuclei EU signal is  \nnormalised to DAPI and log10 scaled. Cells treated with H2O2 (c) or MND (d) and EU-labelled for 30 \nmin. e Schematic of TTchem-seq profiling nascent transcriptome by 4sU incorporation into nascent RNA, \nfragmentation and sequencing of purified labelled RNAs.  f Single-gene view of TT chem-seq data \nbetween untreated controls and 15 min H2O2 treated cells and MND treated cells. g-h Metagene analysis \nof TTchem-seq data between untreated controls and 15 min H2O2 treated cells (g) and MND treated cell \n(h). Lines represent the mean of spike-in normalised read counts between two biological replicates for \na set of protein-encoding genes (non-overlapping ± 2 kb, n = 11,593). i Western blot of total RNAPII \nlevels from chromatin-enriched cellular fractions. Total RNAPII was detected with an antibody raised \nagainst the N-terminal part of RPB1 (D8L4Y). Histone H3 serves as loading control. IIo and IIa denotes \nhyperphosphorylated and unphosphorylated RPB1 CTD respectively. j Schematic of ELCAP -seq \nexperimental design . RNAPII is captured by immunoprecipitation of RPB3 -FLAG, short RNA  \nfragments protected by RNAPII are extracted and sequencing to obtain nucleotide resolution maps of \nRNAPII positions (3’end corresponding to last incorporated nucleotide) . k Heatmaps of ELCAP-seq \nsignal (left: RPKM) alongside TTchem-seq signal (right; read counts normalised to spike-in). The signal \nis shown as the log2 fold -change between H2O2 -treated HEK293 cells and untreated cells. Heatmaps \ncentred on transcription start site and sorted by increasing length of gene regions (−5 kb upstream of \nTSS to 50 kb into the gene body) with dashed lines indicating transcript end sites (TES).  \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\nFig. 2. Oxidative stress arrest RNAPII within the gene body . a Schematic for RNAPII promoter -\nproximal release time points with DRB inhibition and release with and without H 2O2 treatment in \nHEK293 cells. RNAPII complexes are synchronised at promoter-proximal pause site by DRB treatment \nfor 3.5 hours, then released by media washout following either re-inhibition or not and either treated or \nnot with H2O2. b Metagene analysis of TTchem-seq data following 3.5 hours of DRB inhibition between \nno DRB release, 30 mi n released control and 30 min release with 15  min H2O2 treatment of HEK293 \ncells. Data is represented as the average spike-in normalised signal of two biological replicates.  The \narrow represents the change upon H2O2 treatment. c Metagene analysis of TTchem-seq data following 3.5 \nhours of DRB inhibition with DRB release for 15  min and re-inhibition for 15 min with and without \n15min H2O2 treatment. The arrow represents the change upon H2O2 treatment. Data is represented as the \nmean of spike-in normalised TTchem-seq read counts over each segmented bins for the selected genes \nset of protein coding genes and average of two biological replicates.  d-e Single-gene view of TTchem-\nseq data for the PPP2R2A gene for experimental conditions of (b-c) respectively. \n \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\nFig. 3. Arrested RNAPII elongation complexes resume transcription from within the gene body \nduring recovery from oxidative stress . a Metagene analysis of TTchem-seq data for long genes (>90  \nkb, n=2217) following H 2O2 treatment and indicated recovery time points in  HEK293 cells. The line \nrepresents the mean of spike-in normalised read counts from three biological replicates. b Single-gene \nview of TTchem-seq data for FARS2 gene (length = 521 kb)  for experimental conditions of ( a). Arrow \nrepresent distance RNAPII would travel from the TSS with an elongation speed of 2 kb/min or 4 kb/min, \nwithin the 30 min or 1 hour recovery period, respectively. c Schematic of the experimental setup used \nto study RNAPII restart following oxidative stress with or without DRB inhibition.  d Nascent \ntranscription levels (4sU dot blot) after 15 min treatment of HEK293 cells with 1  mM H2O2 with and \nwithout DRB. Staining with methylene blue serves as a loading control. e Metagene analysis of TTchem-\nseq data for long genes (>90 kb, n=2217) of untreated and 1 hour recovery timepoint following H2O2 \ntreatment of HEK293 cells, with and without DRB inhibition. Lines is the mean of spike-in normalised \nread counts between two biological replicates. f Single-gene view of TTchem-seq data for FOXP4 gene \nfor experimental conditions of (c). \n \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\nFig. 4.  NELF mediated RNAPII pausing is altered during the oxidative stress response . a Total \nRNAPII (FLAG IP in HEK293 RPB3-FLAG cells) interactome after 15 min of 1 mM H2O2 treatment. \nA log2  fold-change comparison of H 2O2-treated with untreated control cells is shown relative to \nsignificance as -log2 of p-value (triplicate injections). Are highlighted: RNAPII, NELF and the capping \ncomplex subunits and INTS4 (IP-WB loading control in (c)). b Phosphorylated RPB1 (4H8 IP, HEK293 \ncells) interactome after 15 min of 1 mM H2O2 treatment. A log2 fold-change comparison between H2O2 \ntreated to untreated control cells is shown relative to significance as -log2 of p-value. Are highlighted: \nRNAPII subunits, NELF subunits, members of the capping complex and CPSF2  (IP-WB loading \ncontrol) in (c) are coloured and labelled. c Western blot analysis of FLAG IP (left) and 4H8 IP (right) \nbefore and after H2O2 treatment. Total RNAPII probed with D8L4Y antibody. Phosphorylated RNAPII \nCTD with 4H8. Serine 5 phosphorylated RNAPII CTD with 3E8. d Metagene analysis of ELCAP-seq, \nRPKM signal centred on the TSS for non -overlapping coding genes (n=11 ,593) in untreated control, \nH2O2 treated, and recovery timepoints (30 min, 1 hour and 2 hours). The lines are the average signal of \ntwo biological replicates. Doted vertical lines are the highest RNAPII occupancy distance from TSS  \nupon 30 min recovery and 2 hours recovery . e Heatmap analysis of RPKM ELCAP-seq in RPB3-\n3xFLAG cells treated with H2O2 as in ( d) and NELFE untreated ChIP-seq signal in HEK293. The \nELCAP-seq signal is shown as a log2 fold -change relative to untreated.  NELFE ChIP-seq signal is \nnormalised to IgG input . Top panel shows gene regions (pause −5 kb to pause  +50 kb)  sorted by \nincreasing gene length (pause site genes n= 7 ,595), with dashed lines indicating TES. Middle panel \nshows promoter regions (pause site ± 5 kb) sorted by decreasing NELFE ChIP-seq signal. Bottom panel \nshows promoter regions (pause site ± 0.5 kb) sorted by decreasing NELFE ChIP-seq signal. f Heatmap \nof NELFE ChIP-seq log2 fold-change between H2O2 treated and untreated control and between 1 hour \nrecovery and untreated control, both normalised by ratio to the log2 fold-change of respective treatment \nchanges in total RPB1 (D8L4Y) ChIP-seq in HEK293 cells. Gene regions (± 5 kb pause site genes n = \n7,595) are sorted by decreasing NELFE ChIP-seq signal in untreated condition.  \n \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\nFig. 5. PARylation restricts release of early RNAPII elongation complexes during transcription \nrecovery. a-b Western blot analysis of poly -ADP-ribose levels in HEK293 cells with and without 1 \nhour pre-treatment with PARP inhibitor ( Olaparib) (a) or cells pre -treated for 1 hour with PARPG \ninhibitor (PDD 00017273) (b) and then subsequently treated with 1 mM H2O2 for 15 min and indicated \ntimes following media replenishment. Ponceau S staining serves as total protein loading contro l. c \nNascent transcription levels (4sU dot blot) treated as in ( a) with and without PARPi pre-treatment or \nPARGi. Staining with methylene blue serves as a loading control. d 4sU labelled RNA yields measured \nafter streptavidin pull-down preformed in duplicates of HEK293 cells treated as in (a), with or without \n1 hour PARPi pre-treatment. e-f Metagene analysis of TTchem-seq for long genes (non-overlapping >90 \nkb, n = 2,217) of DMSO treated HEK293 cells (e) or PARPi treated HEK293 cells (f) treated with 1 \nmM H2O2 for 15 min with and without 15 min recovery. Lines is the mean of spike-in normalised read \ncounts between two biological replicates. g Single-gene view of TTchem-seq data for the FOXP4 gene \nfor experimental conditions of (E -F). h Heatmaps of TTchem-seq signal ( normalised to spike -in). The \nsignal is shown as the log2 fold-change between PARPi treated HEK293 cells and DMSO treated cells \nas control. Heatmaps are centred on transcription start site and are sorted by decreasing length of genes \nregions (TSS to +200 kb) with dashed lines indicating TES. \n \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\nFig. 6. Lack of XRCC1 blocks gene body recovery of RNAPII elongation complexes during  \ntranscription recovery. a Nascent transcription levels quantified by 5-Ethynyluridine (EU) labelling \nand single cell quantification by immunofluorescence microscopy (EU-assay). Per cell EU  signal is \nnormalised to DAPI and log 10 scaled. HEK293 WT and XRCC1 knockouts (KO) cells were t reated \nwith 1 mM H2O2 and EU-labelled for 30 min. b Representative images of global nascent transcription \n(EU immunofluorescence) from experiments in ( a) of WT HEK293 and XRCC1 KO cells following \noxidative stress conditions. All image intensities are displayed with same exposure as the u ntreated \ncontrol.  c Heatmaps of TTchem-seq signal (normalised to spike-in) in HEK293 WT cells and XRCC1 KO \ncells. The signal is shown as the log2 fold-change between 1 hour recovery relative to untreated samples \nfor the respective cell lines. Heatmaps are centred on TSS and are sorted by decreasing length of gene \nregions (TSS to +200 kb) with dashed lines indicating TES. d 4sU labelled RNA yield measured after \nstreptavidin pull-down in HEK293 cells (WT), XRCC1 KO (average for two independently generated \nKO cell lines), HEK293 treated with PARPi (WT + PARPi) and XRCC1-KO treated with PARPi \n(XRCC1 KO + PARPi). All samples were performed in duplicates. e-f Metagene analysis of TTchem-seq \ndata for 1 hour recovery for long protein coding genes (>90 kb, n=2,217). Metagene in (e) is centred on \nTSS (-2 kb to TSS to +80 kb) while metagene in (f) is scaled on transcription start site and transcript \nend site (-2 kb to TSS  to TES +2 kb). Lines is the mean of  spike-in normalised read counts between  \ntwo biological replicates. g Single-gene view of TTchem-seq data for the CERS6 gene for experimental \nconditions of (e-f). h Heatmaps of TTchem-seq signal (normalised to spike-in). The signal is shown as \nthe log2 fold-change between WT-DMSO control and PARPi treated XRCC1-KO cells. Heatmaps are \ncentred at the transcription start site (TSS + 200 kb) and sorted based on gene lengths. \n \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\nFig. 7. Lack of OGG1 activity blocks gene body recovery of RNAPII elongation complexes during \ntranscription recovery. a Nascent transcription levels quantified by 5-Ethynyluridine (EU) labelling \nand single cell quantification by immunofluorescence microscopy (EU-assay). Per cell EU  signal is \nnormalised to DAPI and log 10 scaled. HEK293 WT and OGG1i treated HEK293 cells were t reated \nwith 1 mM H2O2 and EU-labelled for 30 min. b Representative images of global nascent transcription \n(EU immunofluorescence) from experiments in (a) of WT HEK293 and OGG1i treated HEK293 cells \nfollowing oxidative stress conditions. All image intensities are displayed with same exposure as the \nuntreated control.  c Heatmaps of TTchem-seq signal (normalised to spike-in) in HEK293 WT cells and \nOGG1i treated HEK293 cells. The signal is shown as the log2 fold -change between 1 hour recovery \nrelative to untreated samples for the respective cell lines. Heatmaps are centred on TSS and are sorted \nby decreasing length of gene regions (TSS to +200 kb) with dashed lines indicating TES. d 4sU labelled \nRNA yield measured after streptavidin pull-down in HEK293 cells (WT), HEK293 treated with OGG1i, \nHEK293 treated with PARPi (WT + PARPi) and OGG1i treated HEK293 with PARPi (OGG1i + \nPARPi). All samples were performed  in duplicates. e-f Metagene analysis of TT chem-seq signal for 1 \nhour recovery for long protein coding genes (>90 kb, n=2,217). Metagene in (e) is centred on TSS (-2 \nkb to TSS to +80 kb) while metagene in (f) is scaled on transcription start site and transcript end site (-\n2 kb to TSS to TES +2 kb). Lines is the mean of spike-in normalised read counts between two biological \nreplicates. g Single-gene view of TTchem-seq data for the PAM gene for experimental conditions of (e-\nf). h Heatmaps of TTchem-seq signal ( normalised to spike -in). The signal is shown as the log2 fold-\nchange between WT-DMSO control and PARPi treated OGG1i treated HEK293 cells. Heatmaps are \ncentred at the transcription start site (TSS + 200 kb) and sorted based on gene lengths. i Model of the \ntranscriptional response to oxidative damage. In WT cells (left) oxidative damage induced by hydrogen \nperoxide or menadione results in rapid RNAPII arrest, NELF-mediated promoter proximal pausing and \nPARylation. As transcription recovers RNAPII elongation resumes from within the gene body. NELF \nis disassociated from early RNAPII elongation complexes and released into the gene body due to de-\nPARylation. In XRCC1 KOs or upon OGGi RNAPII elongation block is maintained (right top panel), \nPARPi treatment results in forced release of early RNAPII elongation complexes (right middle panel), \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint \n\nwhereas PARPi in XRCC1 KOs or OGG1i leads to forced release of early RNAPII elongation \ncomplexes with a sustained elongation block. \n \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 6, 2025. ; https://doi.org/10.1101/2025.11.06.686939doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}