Arp2/3 and Type-I myosins control chromosome mobility and end-resection at double-strand breaks in S. cerevisiae

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Zhou, Marissa Ashton, Yiyang Jiang, Neha Arora, Kevin Clark, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5125308/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Aug, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Using budding yeast, S cerevisiae , we show that Arp2/3 actin branching complex has an evolutionarily conserved role in promoting chromosome mobility of double-strand breaks (DSBs). Disrupting Arp2/3 activity after DSB induction, by drug treatment with CK-666 or auxin-induced degron depletion of nucleation promoting factors Las17 WASP or the type-1 myosins (Myo3 and Myo5), markedly reduced the radius of confinement of DSBs. Arp2/3 was found to be required to initiate and maintain 5’to 3’ resection of DSB ends. Conversely, inhibiting Exo1 and Dna2- dependent long-range resection or deleting FUN30 impaired the mobility of the DSB, while overexpression of Exo1 suppressed resection inhibition by CK-666. Inactivation of Arp2/3 before DSB induction triggered a shortened checkpoint arrest through the Tel1 ATM /Mre11 (TM) checkpoint, while inactivation of Arp2/3 after DSB induction caused cells to escape arrest prematurely. Shortened checkpoint arrest correlated with a reduction in repair by interchromosomal homologous recombination. These results suggest that Arp2/3 regulation plays an unanticipated role in the regulation of processing of DSB ends that is correlated with an increase in DSB mobility and DSB repair. Biological sciences/Molecular biology/DNA damage and repair/Double-strand DNA breaks Biological sciences/Molecular biology/DNA damage and repair/Homologous recombination Biological sciences/Molecular biology/DNA damage and repair/DNA damage response double-strand break (DSB) mobility DSB repair Arp2/3 actin myosin Las17WASP homology search DSB end resection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Repair of double-strand breaks (DSBs) is required to maintain genome integrity. Homologous recombination (HR) requires that the ends of the DSB find a homologous donor sequence to use as a template for repair (reviewed in [ 1 ]). In budding yeast, a DSB causes an increase in chromatin mobility near a DSB break site [ 2 , 3 ], which is thought to facilitate the Rad51-mediated search for homology by increasing the volume that the DSB ends explore within the nucleus, defined as the radius of confinement (Rc) [ 4 ]. While processes such as the activation of the Mec1 ATR -dependent DNA damage checkpoint, the phosphorylation of histone H2A, and recruitment of repair proteins to the break have been shown to be required for DSB-induced chromatin mobility [ 2 , 3 ], there is still no mechanism that explains how a DSB causes increased chromatin mobility or how changes in mobility affect DSB repair (reviewed in [ 4 , 5 ]). Several studies have suggested that actin branching is directly involved in repairing double-strand breaks. Recently it has been shown in mammalian cells and Drosophila that inactivation of the actin nucleator Arp2/3 by the drug CK-666 lowers DSB mobility [ 6 , 7 ]. Treatment of Drosophila cells with CK-666 or RNAi treatment of nuclear myosin proteins impaired the directed movement of damaged DNA in heterochromatin to the nuclear periphery [ 6 ]. In addition, CK-666 caused an apparent reduction in the amount of single-stranded DNA (ssDNA) formed at DSB ends [ 7 ]. A proteomics study in Xenopus egg extracts has shown that all seven-subunits of Arp2/3 and actin are enriched at DSBs [ 7 ]. The branching of polymerized actin is dependent on the Arp2/3 complex and a set of nucleation promoting factors (NPF) (reviewed by [ 8 ]. The Arp2/3 actin nucleator Wiskott-Aldrich syndrome protein (WASP) [ 9 , 10 ] has been shown to affect the loading of the single-strand binding protein RPA onto single-stranded DNA in mammalian cells [ 11 ] and mutations in Las17, the budding yeast homologue for WASP, was shown to have hypersensitivity to the DNA damaging agents hydroxyurea (HU) and methyl methanesulfonate (MMS) [ 11 ]. Las17 mutants accumulated more Rad52 foci when exposed to HU or MMS, suggesting a deficiency in repair by HR [ 11 ]. Here we have investigated the role of Arp2/3 and its associated NPFs in S. cerevisiae in more detail. In budding yeast, Arp2/3 and the NPFs Las17 and the type-I myosins (Myo3 and Myo5) (reviewed in [ 12 ]) are primarily found in cortical actin patches and are required for clathrin-mediated endocytosis (CME) [ 8 ]. These factors are well-studied in endocytosis [ 12 ], but their roles in other cellular processes remain poorly defined. Here we have used Ddc2-GFP to directly label DSBs [ 13 ] and mean-square displacement (MSD) analysis (reviewed in [ 14 ]) to track and measure changes in mobility of an irreparable site-specific DSB at the MAT locus on chromosome III. We found that blocking Arp2/3 activity, either through the drug CK-666 or through a deletion/mutation of NPFs, lowered DSB mobility. Moreover, 5’ to 3’ resection was reduced when Arp2/3 was inactivated after DSB formation. When Arp2/3 activity is blocked before DSB induction, the initiation of resection was severely impaired. Additionally, we show that altering the rate of resection, through deleting the chromatin remodeler Fun30 or inactivating the Exo1 and Dna2 nucleases responsible for long-range resection, lowered DSB mobility. Altogether our study shows that Arp2/3 has an evolutionarily conserved role in regulating both DSB mobility and DSB end-resection, and that directly impairing the rate of resection prevents the increased motion of DSB ends. Results Labeling and tracking the mobility of double-stranded breaks To generate, track, and quantify the mobility of a single-irreparable DSB we modified the well-characterized strain JKM179, in which a single site-specific cut in the MAT locus on chromosome III is induced by a galactose-regulated HO-endonuclease gene (Gal-HO) [ 15 ]. To visualize and track the DSB, we monitored the DNA damage checkpoint protein Ddc2 (Ddc2-GFP) which we have previously shown forms a damage-dependent GFP focus in ~ 80% of cells 3 h after adding galactose [ 13 ]. An mCherry-tagged spindle pole body protein Spc42 (Spc42-mCherry) was used as a fiducial marker to account for random nuclear motion. Cutting by Gal-HO is highly efficient with nearly 90% of cells being cleaved 1 h after galactose addition [ 15 – 17 ]. In these strains the homologous HML and HMR donors are deleted, thus preventing repair of MAT by HR. When HO is continually expressed, repair of the DSB by altering the cleavage site via nonhomologous end-joining occurs in only 0.2% of cells [ 18 ]. We measured DSB mobility 3 h after adding galactose and quantified the mobility of the DSB using mean-squared displacement (MSD) analysis (see Methods for details) (Fig. 1 A-B). Blocking Arp2/3 activity lowers DSB mobility To test whether Arp2/3 activity has a conserved role in DSB mobility in budding yeast, we added 10µM CK-666 to block Arp2/3 activity 20 min before imaging (160 min after adding galactose) (Fig. 1 C). Addition of CK-666 significantly lowered the radius of confinement (Rc) of the DSB from 0.92 µm to 0.69 µm (Fig. 1 B and Table S1 ). Addition of CK-666 did not affect the mobility of the spindle pole bodies relative to the bud neck ( Figure S1 A-B, Table S1 ), suggesting that the changes seen in DSB mobility when cells are treated with CK-666 were due to the changes in the behavior of the DSB. Since Arp2/3 is primarily known as a component of endocytosis in budding yeast, we asked whether the change in DSB mobility was due to impaired endocytosis. Deletion of SLA2 , a key component of endocytosis [ 19 ], did not affect the behavior of the DSB (Fig. 1 D-E and Table S1 ). This result suggests that endocytosis is not required for DSB mobility and that the role of Arp2/3 in DSB mobility is independent of its role in endocytosis. Arp2/3 affects changes in local and global chromatin mobility in response to a DSB While the direct labeling of DSBs with Ddc2-GFP is suitable for tracking the mobility of DSBs, it cannot be used to establish a basal level of chromatin mobility in the absence of a DSB. To test whether Arp2/3 is required for increasing chromatin mobility near a DSB, we induced an irreparable DSB at MAT in a derivative of strain JKM179 that expresses the GFP-LacI protein that binds to a lacO array inserted 4.4 kb away from the MAT locus [ 20 ] (Fig. 2 A). As previously reported [ 20 ], we found that inducing a DSB caused an increase in local chromatin mobility near the DSB site: R C 0.63 µm (uncut) and 1.1 µm (cut) (Fig. 1 B, G and Table S1 ). CK-666 did not significantly affect basal levels of chromatin mobility: Rc uncut (0.63 µm) and uncut + CK-666 (0.54 µm) (Fig. 1 B, G and Table S1 ); however, treatment with CK-666 either 20 min before (Fig. 2 C) or 160 min after (Fig. 2 E) DSB induction resulted in no increase in DSB mobility compared with the uncut condition, monitored at 180 min (Fig. 2 D, F-G and Table S1 ). Thus, CK-666 impairs DSB-induced chromatin mobility. Nucleation promoting factors are required DSB-induced increase in mobility In humans, Arp2/3 requires the NPF WASP to nucleate new branches of actin [ 9 , 10 ]; Las17 is the budding yeast homologue of WASP (Fig. 3 A) [ 21 ]. To test whether Las17 was required for DSB mobility, we added an auxin-inducible degron (AID) to the C-terminus of Las17 (Las17-AID) [ 22 , 23 ]. Adding 1mM auxin (IAA) caused degradation of Lad17-AID within 1h (Fig. 3 B). The AID-tag did not affect the MSD curve of Las17-AID, but when IAA was added 2 h after Gal-HO induction, the mobility of the DSB measured at 3 h dropped: Rc WT (0.83 µm), Las17-AID (0.77 µm), and Las17-AID + IAA (0.49 µm) (Fig. 3 C-D, Table S1 ). To further characterize the role of Las17, we deleted functional domains that are responsible for Arp2/3 activation. Deletion of the acidic patch (CA) domain ( las17-CA∆ ), which directly binds to Arp2/3 to activate Arp2/3 [ 24 ], did not result in a significant change in the mobility of the DSB: Rc WT (0.91 µm) and las17-CA∆ (0.90 µm) (Fig. 3 E-F, Table S1 ). However, additional deletion of the WH2 domain, which brings monomeric actin to Arp2/3, along with the CA domain ( las17-WH2-CA Δ) lowered the mobility of the DSBs: R C las17-WH2-CA Δ (0.53 µm) (Fig. 3 E-F, Table S1 ). This suggests that Las17 in its role as an Arp2/3 actin nucleator is required for DSB mobility. The type-I myosins Myo3 and Myo5 play a role in DSB mobility Type-I myosins Myo3 and Myo5 promote actin nucleation by Arp2/3 during endocytosis in budding yeast [ 25 ] and depletion of Myo1a and Myo1b in Drosophilia increases sensitivity to ionizing radiation [ 6 ]. We asked if Myo3 or Myo5 have a role in DSB mobility in budding yeast. We found that a single deletion of MYO3 or MYO5 was sufficient to lower the mobility of the DSB: Rc WT (0.83 µm), myo3∆ (0.60 µm), and myo5∆ (0.58 µm) (Fig. 4 A, C and Table S1 ). The effect of deleting either type-I myosin was different from what is seen in endocytosis, where Myo3 and Myo5 are functionally redundant [ 26 ]. We then asked whether a second copy of one type-I myosin, added to a deletion of the other, would restore DSB mobility. We integrated a second copy of MYO5 at URA3 on chromosome V, under control of its own promoter, in a myo3 Δ background and found that there was no significant difference in the Rc between the WT (0.83 µm) and the myo3 Δ Myo5 (+ Myo5) (0.80 µm) strains. (Fig. 4 B-C, Table S1 ). To determine which functional domains of Myo5 are required for DSB mobility, we integrated a series of MYO5 domain deletion plasmids with their endogenous promotors [ 25 ] into a myo5 D strain. In the presence of Myo3, we found that there was no significant difference in the R C between the wildtype strain and a deletion of the Myo5 motor-domain, TH1, or TH2 domains: WT (0.89 µm), myo5-motorΔ (0.86µm), myo5-TH1Δ (0.85 µm), and myo5-TH2Δ (0.96 µm) ( Figure S2A-C, E and Table S1 ). However, a deletion of the SH3 domain ( myo5-SH3Δ ) did not restore the mobility of the DSBs back to wildtype levels; R C WT (0.89 µm) and myo5-SH3Δ (0.62 µm) ( Figure S2D-E, Table S1 ). Because a double deletion of MYO3 and MYO5 is synthetically lethal in most backgrounds [ 27 , 28 ], we added an AID tag to Myo5 (Myo5-AID) in a myo3 D strain. In a MYO3 strain, the addition of the AID tag to Myo5 did not affect the mobility of the DSB and adding IAA 2 h after DSB induction reduced mobility, similar to the MYO5 deletion. ( Figure S3, Table S1 ). Unexpectedly, in the myo3∆ Myo5-AID strain, treatment with IAA increased the mobility of the DSB to near WT levels: Rc WT (0.81 µm), myo3∆ Myo5-AID (0.6 µm), and myo3∆ Myo5-AID + IAA (0. 81 µm) (Fig. 4 D-E, Table S1 ). Since Las17 has a WH2 domain and acidic (CA) patch, it is possible that the increase in DSB mobility when both Myo3 and Myo5 are absent could be due to the Arp2/3 actin nucleation by Las17. To test this, we deleted the WH2 and CA domains of Las17 ( las17-WH2-CA Δ) in a myo3 Δ Myo5-AID background and measured the mobility of the DSB ± IAA as described above. We found that the las17 mutant prevented the restoration of Rc to WT levels that was seen when both Myo3 and Myo5 are inactivated: Rc WT (0.99 µm), myo3 Δ Myo5-AID las17-WH2-CA Δ (0.57 µm), and myo3 Δ Myo5-AID las17-WH2-CA Δ + IAA (0.58 µm) ( Figure S4, Table S1 ). Together these results suggest that Myo3 and Myo5 in their role as Arp2/3 actin nucleators play a role in DSB mobility and Las17 can compensate for the loss of both Myo3 and Myo5 to maintain near WT levels of DSB mobility. Arp2/3 and myosins are required for damage-dependent focus formation with Ddc2-GFP or Rad51-GFP Ddc2-GFP and Rad51-GFP form damage-dependent foci in ≥ 80% of cells 3 h after DSB induction [ 13 ]. However, when Arp2/3 activity was blocked by CK-666 before DSB induction, we observed that Ddc2-GFP and Rad51-GFP formed foci in fewer than 15% of cells ( Figure S5A-B ). Similar inhibition of the DSB-induced foci was found in a deletion of VRP1 , or when IAA was added to Las17-AID or myo3Δ Myo5-AID cultures. Deletion of KU70 did not restore damage-dependent Ddc2-GFP foci formation in CK-666 treated cells ( Figure S5C ). This finding raised the possibility that blocking Arp2/3 might interfere with either Gal-HO cutting or with long-range resection. By using a pair of PCR primers that flank the HO-cut site in the MAT locus ( Figure S5A ) we showed that Gal-HO cutting was unaffected in Las17-AID or myo3∆ Myo5-AID mutants treated 1 h before DSB induction with IAA ( Figure S5B-D ). Arp2/3 and type-I myosins are required for the initiation and maintenance of resection Since Gal-HO cutting is normal, blocking Arp2/3 activity might interfere with 5’ to 3’ resection of the DSB. We first monitored the disappearance of GFP-LacI/ lacO foci in a strain where the lacO array was inserted 4.4 kb away from the HO cleavage site at MAT (Fig. 2 A). As resection erodes the lacO array, GFP-LacI will lose its binding sites and the GFP-LacI/ lacO focus will vanish. Within 2 h after DSB induction there was a steady drop in the percentage of cells with a GFP-LacI focus (Fig. 5 A). However, addition of CK-666 1 h before HO induction prevented the loss of the GFP-LacI focus. Resection in cells treated with CK-666 after 1 h after DSB induction was also significantly impaired. We used a restriction enzyme-based qPCR resection assay to measure the generation of ssDNA around the HO-cut site itself [ 29 , 30 ]. qPCR analysis was done using a series of primers flanking STY1 cut sites at different distances from the HO-cut site in the MAT locus (0.7 kb, 5 kb, and 10 kb) (Fig. 5 B). As resection converts dsDNA to ssDNA, Sty I cleavage sites are lost and qPCR threshold signal is detected. We measured resection in Las17-AID, myo3∆ , myo5∆ , and myo3Δ Myo5-AID mutants. IAA was added either 1 h before or 2 h after adding galactose. Las17-AID without IAA had a similar resection profile as the wildtype; however, when IAA was added either before or after galactose, long range resection was severely impaired (Fig. 5 C) suggesting that Las17 is required for the initiation and maintenance of resection. Single deletions of MYO3 or MYO5 did not greatly affect resection (Fig. 5 D). Resection in the myo3∆ Myo5-AID mutant without IAA was comparable to resection in the WT, as expected from the myo3∆ resection data (Fig. 5 D-E); but when IAA was added to myo3∆ Myo5-AID 1 h before DSB induction, there was little to no resection up to 6 h after DSB induction (Fig. 5 E). Finally, when IAA was added 2 h after DSB induction, resection in myo3 D Myo5-AID appeared to be greatly inhibited within an hour of degrading Myo5 (Fig. 5 E). Together these data suggests that Arp2/3 and type-I myosins have a role both in the initiation of resection and in its maintenance. Changes in resection lower the mobility of DSBs Since resection was affected by changes in Arp2/3 activity, we asked whether there was a correlation between the rate of resection and DSB mobility. Previously we and others have shown that a deletion of the chromatin remodeler FUN30 greatly reduced Exo1 and Sgs1/Dna2-dependent long-range resection [ 31 – 33 ]. Although resection through the 0.7 kb site was not affected in fun30∆ , long-range resection past the 5 and 10 kb sites was markedly reduced (Fig. 6 A). MSD analysis of a DSB showed the R C of fun30 D (0.5 µm) was significantly lower than the WT (0.8 µm) (Fig. 6 B-C, Table S1 ). Long-range resection requires both the exonuclease Exo1 and the helicase/endonuclease DNA2-Top3-Rmi1-Sgs1 complex [ 34 , 35 ]. Since the rate of resection is not affected by deleting either EXO1 or SGS1 alone [ 33 ], we blocked long range resection by deleting EXO1 and degrading Dna2-AID. There was no difference in DSB mobility between the wildtype and exo1 D Dna2-AID, but when IAA was added 2 h after DSB induction the mobility of the DSB measured at 3 h was reduced: Rc WT (0.8 µm), exo1 Δ DNA2-AID (0.8 µm), and exo1 Δ DNA2-AID + IAA (0.5 µm). (Fig. 6 D-E, Table S1 ). To test whether increasing resection affected DSB mobility, we measured mobility in a strain with a second copy of EXO1 expressed under control of a galactose promoter (pGal: EXO1 ) at the LEU2 locus [ 36 ]. We found that there was no difference between the Rc in WT and when Exo1 was overexpressed (Fig. 6 F, H and Table S1 ) even though the rate of resection was increased (Fig. 6 I). However, pGal:: EXO1 suppressed the reduction in Rc from CK-666 (Fig. 6 F-H, Table S1 ), but did not rescue Ddc2-GFP focus formation in cells treated with CK-666 before DSB induction ( Figure S5D ). Taken together, continuous resection is required for the high mobility of DSBs and overexpression of Exo1 can overcome the reduced rate of resection caused by CK-666 but could not prevent the block to the initiation of resection. Activation of the TM checkpoint in response to blocking Arp2/3 activity When a DSB is detected, the DNA damage checkpoint (DDC) is activated to halt cell cycle progression and allow cells a chance to repair the DSB before proceeding through mitosis [ 37 , 38 ]. In budding yeast, activation of the DDC is mainly controlled through the effector kinase Mec1 ATR with a minor contribution by the Tel1 ATM kinase [ 39 – 42 ]. Mec1 is recruited to DSBs by its binding partner Ddc2 ATRIP , which binds to RPA loaded on ssDNA [ 43 , 44 ]. Arrest by the DDC is typically monitored by cell morphology, as cells shift towards a large-budded G 2 /M state, and by Western blot analysis of phosphorylation of the Mec1/Tel1 target, Rad53 [ 45 ]. We hypothesized that preventing resection - by blocking Arp2/3 activity prior to HO induction - should interfere with cell cycle arrest through the DDC. Typically, a cell with a single irreparable DSB will arrest for 12–15 h before escaping arrest through a process known as adaptation [ 46 ]. Here we find that when Arp2/3 activity was blocked before DSB induction, either by drug treatment with CK-666 or degradation of Las17-AID with IAA (Fig. 7 A), G 2 /M arrest was shortened to 4 h (Fig. 7 D-G). Deletion of either Myo3 or Myo5 did not shorten checkpoint arrest (Fig. 7 D-E). While Mec1 is primarily responsible for checkpoint arrest by the DNA damage checkpoint, the Tel1-Mre11 (TM) checkpoint is an alternative checkpoint response in response to a DSB, notably in mec1 D cells in which initial resection by the Mre11 complex is impaired [ 47 ]. We confirmed that the short checkpoint response we observed was attributable to the TM checkpoint, as tel1 D and mre11 D strains treated with CK-666 before DSB induction did not exhibit a checkpoint response (Fig. 7 B-C). A mre11Δ Las17-AID double mutant treated with auxin also showed little or no G 2 /M arrest and western blot analysis showed that Rad53 was not phosphorylated (Fig. 7 H-I). Since degrading Las17 after DSB induction impaired further resection, we assayed whether Arp2/3 is required to maintain checkpoint arrest by adding CK-666 to our wildtype strain or IAA to Las17-AID 2 h after adding galactose and monitored for changes in cell morphology (Fig. 7 A). We found that treatment with CK-666 or degrading Las17-AID resulted in cells escaping G 2 /M arrest early despite Rad53 remaining hyperphosphorylated (Fig. 7 J-K). These results show that the shortened DSB-induced checkpoint arrest was dependent on the TM checkpoint which was activated when resection was impaired by inhibiting Arp2/3 or its associated factors. DSB Mobility and Repair by Gene Conversion If DSB-induced chromatin mobility facilitates homology search, then lowering the mobility of DSBs might impact the repair efficiency carried out by gene conversion (GC). To study how changes in chromatin mobility affect the efficiency of DSB repair, we modified the well-characterized strain YJK17 which has an ectopic, mutated copy of MAT ( MAT a-inc ) that cannot be cut by the HO-endonuclease and which serves as a donor to repair the cleavage at MAT α by interchromosomal gene conversion (Fig. 8 A) [ 33 , 48 ]. With this strain we could generate and monitor the repair of a single DSB event. Deletion of MYO3 , MYO5 , or MRE11 did not impact repair; however Las17-AID showed a significant decrease in viability from YP-Gal (79%) and YP-Gal + IAA (46%) plates (Fig. 8 B). An mre11Δ Las17-AID double mutant showed an even more significant drop in viability (65% on YP-Gal and 10% on YP-Gal + IAA) (Fig. 8 B). Las17-AID survivors from the YP-Gal and YP-Gal + IAA plates grew normally on YPD and YP-Gal plates ( Figure S7 ) as expected. To further test the effects of mobility-deficient mutants on GC, we deleted MYO5 and modified Las17 in a series of strains we had previously characterized with repair efficiencies ranging from 46–9% [ 49 ]. The differences in repair efficiencies reflect the contact probability between the DSB site and the LEU2 donor sequence. As with YJK17, we found that myo5 D did not affect repair efficiency (Fig. 8 D) but Las17-AID + IAA showed a significant decrease in the percentage of survivors in all 4 strains (Fig. 8 E). Since degradation of Las17 showed reduced resection, we wanted to know how this would affect repair by single-strand annealing (SSA) using the previously characterized strain YMV2, which has an HO cut-site inserted in LEU2 on chromosome 3 ( leu2-cs ) and a 1.3 kb fragment of the 3’ end of LEU2 (U2) inserted 30 kb upstream of leu2-cs (Fig. 8 F) [ 50 ]. The DSB can be repaired both by Rad51-dependent break-induced replication and by Rad51-independent SSA [ 33 ]. Degradation of Las17 reduced the survival rate from 86–63% (Fig. 8 G). Deletion of RAD51 did not affect the repair efficiency, 85% (Fig. 8 G). Yet when Las17 was degraded in the rad51Δ background, repair efficiency dropped to 36% (Fig. 8 G). This suggests that Las17 and Arp2/3 have a role in repair by SSA and have much less effect on BIR, where much less resection is required. Discussion When a cell commits to repair a DSB through HR, there is an increase in chromatin mobility near the DSB [ 2 , 3 , 20 ]. While it is thought that increased chromatin mobility near a DSB facilitates homology search, it is unclear what role DSB-induced chromatin mobility has in DSB repair by HR. In this study we investigated the role of the Arp2/3 actin nucleator in DSB mobility and repair by HR. Here we show that Arp2/3, previously shown to be required for DSB mobility in Drosophila and mammalian cells [ 6 , 7 ], has a conserved role in DSB mobility in budding yeast and that this process is separate from Arp2/3’s previously well-characterized function in endocytosis [ 51 ]. It appears that Arp2/3 is required at 2 different steps during homology-based repair: first, for the initiation, and then for the maintenance of long-range resection and, perhaps consequently, for the increased mobility of the DSB during homology searching. Long-range resection was not seen when Arp2/3 activity was blocked before DSB-induction, either through treatment with the Arp2/3 inhibitor CK-666, deletion of VRP1 , degradation of Las17 WASP , or when both type-I myosins, Myo3 and Myo5, were not present. Unlike a SAE2 deletion mutant, which has been reported to have delayed DSB-induced chromatin mobility [ 3 ] and delayed resection [ 52 ], we saw little or no resection up to 6 h after inducing an HO break when Arp2/3 was blocked prior to DSB induction. In previous studies, deletion of YKU70/80 allowed Exo1-dependent resection of DSB ends in G1-arrested cells [ 17 ] and overexpression of YKU70/80 interfered with Mre11 association with DSB ends and acted as a barrier for resection [ 53 ]. Here we show that a deleting YKU70 did not overcome the CK-666 block to forming Ddc2-GFP foci, suggesting that Arp2/3 does not affect the residence of yKu70/80 at DSB ends. The connection between increased chromatin mobility and end-resection is complex. As in mammalian cells, slowing down resection reduces the mobility of DSB ends, though whether the same factors are involved remains unclear. In mammals, inhibition of the Mre11 by mirin reduced mobility [ 7 ], while in budding yeast inhibition of the Mre11 complex by deletion of SAE2 only delays resection and the increase in Rc [ 3 , 52 ]. Here we found that Rc was reduced by impairing long-range resection, either by deleting the chromatin remodeler FUN30 or by inactivation of both Exo1 and Dna2. Thus, a defect in long-range end-resection is sufficient to impair damage-induced chromatin mobility. We also found that EXO1 overexpression suppressed the reduction in Rc caused by adding CK-666, but overexpression of EXO1 was not able to bypass the block in initiating resection when Arp2/3 was inhibited prior to DSB induction. We were unable to test DSB mobility in a triple deletion mutant of the primary resection proteins because an mre11-nd (nuclease dead) exo1 sgs1Δ deletion is inviable [ 35 ]; but it would appear that inactivating Arp2/3 in an mre11 D background eliminates both long- and short-range resection and thus recombination. These findings confirm and extend the analysis of the role of Arp2/3 in mammalian cells, where treatment with CK-666 impaired both mobility and end-resection in human cells [ 7 ]. Detection of a DSB triggers cell cycle arrest through the DNA damage checkpoint (DDC) for 12–15 h [ 46 ]. We found that when Apr2/3 was blocked, cells experienced a 4 h checkpoint which was dependent on the Tel1-Mre11 (TM) checkpoint which arrests cells in response to unresected DSB ends [ 54 , 55 ]. Disruption of both the TM checkpoint and Arp2/3 activity prevented cells from entering DSB-induced checkpoint arrest as evidenced by the lack of cells accumulating in G 2 /M arrest and the lack of phosphorylation of Rad53. Arp2/3 was also required to maintain checkpoint arrest as cells began to escape arrest, despite continued Rad53 phosphorylation. Impaired Mec1-mediated checkpoint arrest in budding yeast is consistent with the lower ATR signaling reported in WASP-deficient human cells [ 11 ]. Of the known Arp2/3 actin nucleation promoting factors in budding yeast, we focused on Las17 WASP and the type-I myosins Myo3 and Myo5. In humans WASP has been shown to bind to RPA and is implicated in loading RPA onto ssDNA in response to a DSB as mutations in WASP were shown to have less RPA-bound DNA when exposed to the topoisomerase I inhibitor camptothecin (CPT), which causes replication fork collapse [ 7 ]. We show that the highly conserved C-terminal region across WASP orthologs (WH2 and CA domains) of Las17 is required for DSB mobility. Human WASP binds to RPA, through this highly conserved region and mediates RPA binding to ssDNA [ 11 ]. Impaired RPA binding to ssDNA would prevent Ddc2 ATRIP -mediated Mec1 activation of the DDC. Budding yeast Myo3 and Myo5 are functionally redundant in endocytosis and deletion of either Myo3 or Myo5 did not affect resection, checkpoint arrest, or repair by gene conversion. However, a deletion of either myosin protein lowered DSB mobility and the insertion of a second copy of Myo5 with its endogenous promotor into a myo3 deletion mutant restored DSB mobility to wildtype levels. Through domain deletion mutants of MYO5 , we found that the SH3 domain of Myo5, which mediates interactions with Vrp1 [ 56 , 57 ], is required for wildtype levels of DSB mobility. Since SH3 domains mediate a wide range of protein-protein interactions, the loss of either type-I myosin might result in an imbalance of interactions required for DSB mobility and the deletion of MYO3 and MYO5 might restore that balance. The loss of both type-I myosins after DSB induction brought DSB mobility back to wildtype levels in a Las17-dependent manner. Deletion of VRP1 impaired the initiation of resection. Since Vrp1 brings Myo3 and Myo5 to Arp2/3 [ 56 ], the deletion of VRP1 would likely be similar to when both Myo3 and Myo5 are degraded. Changes in DSB mobility did not necessarily affect overall DSB repair efficiency through HR. While the degradation of Las17 negatively affected the repair efficiency of DSBs through GC and SSA, deletion of either MYO3 or MYO5 did not affect the repair efficiency of DSBs, even when the donor location was moved to unfavorable locations. This difference in repair efficiencies among these mobility deficient mutants might be due to the length of G2/M arrest imposed by the DDC. Since myo3Δ and myo5Δ mutants did not affect the length of checkpoint arrest, these mutants had the same amount of time to repair the break as the wildtype. The shortened checkpoint arrest when Las17 was degraded might have triggered an early adaptation response. By using single particle tracking and MSD analysis of a single DSB in the MAT locus on chromosome III in budding yeast, we were able to show that Arp2/3 has a conserved role the DNA damage response and plays an important part in the initiation and facilitation of the resection of DSB ends. The lower DSB mobility that we observe when Arp2/3 activity is blocked, either through drug treatment or degradation of Las17 WASP , is likely linked to a lower rate of resection, as DSBs in other resection mutants, such as a FUN30 deletion or an exo1 dna2 double deletion, are less mobile than their wildtype counterparts. The rate of resection is not the only determinant of DSB mobility as a deletion of either Myo3 or Myo5 is sufficient to lower DSB mobility but did not affect the rate of resection. Preventing resection triggered a shortened checkpoint arrest through the TM checkpoint and stopping continued resection allowed cells to escape checkpoint arrest early. Methods KEY RESOURCES TABLE Refer to excel file for tables Methods Details Strain and plasmid construction All strains are derived from JKM179 [15] which is a well characterized strain with the HML and HMR donor domains deleted. Ddc2 and Rad51 were GFP-tagged with a 13 amino acid linker GGSGGSRIPGLIN-eGFP as previously described [13]. Ddc2-GGSGGSRIPGLIN-eGFP and Rad51-GGSGGSRIPGLIN-eGFP are referred to as Ddc2-GFP and Rad51-GFP respectively in this study. All AID-tagged mutants strains were derived from a modified version of JKM179 expressing osTIR1 at URA3 created by cutting pNHK53 [23] with Stu I and integrating into the genome. For degron tagged derivatives, PCR products were generated with mixed oligoes with homology to the C-terminal end and plasmid pJH2892 or pJH2899 [22] to create an 9xMyc-AID (AID) inserts with the KAN or NAT marker. Myo5-domain deletion plasmids [25] were digested with BamHI/SacI to excise a linear fragment for genome integration. Deletion of ORFs, myo5 mutants, and AID tags were introduced with the one step PCR homology cassette amplification and the standard yeast transformation method [58]. Las17 domain deletion mutants were created by CRISPR/Cas9 as previously described [59] with material listed in supplemental tables. Transformants were verified by PCR and western blot. Strains are listed in Table S3 . Growth conditions Strains containing degron fusions and galactose-inducible HO were cultured using standard procedures. Briefly, a single colony grown on a YPD plate was inoculated in 5ml YP-lactate for ~15 hours overnight at 30° C with agitation. The following day, 500-100ml of YP-lactate was inoculated with the starter culture so that the cell density reached OD 600 = 0.5 the following day after overnight growth at 30 ° C with agitation. Once cultures reached the appropriate density, HO expression was induced by addition of 20% galactose to 2% vol/vol final concentration. For auxin treatment, indole-3-acetic acid (IAA) was resuspended in ethanol to a working concentration of 500 mM. For AID degradation cultures were split at the indicated timepoints to add auxin, IAA was added to one culture to a final concentration of 1mM either 1 h before or 2 h after adding galactose. The equivalent volume of 100% ethanol was added to the second culture. Cells were harvested at the indicated timepoint by centrifugation at 3000x for 3 minutes and prepared for microscopy, resection assays, and western blot analysis. Cells prepared for microscopy were washed 3 times with a Leu- media. Plating assays The efficiency of DSB repair by homologous recombination or single-strand annealing was determined as previously described for YJK17 [48]. Briefly, cells were selected from a single colony on YPD plates and grown overnight in 5ml of YEP-lactate. Cells were diluted to OD 600 = 0.2 and allowed to grow until OD 600 = 0.5-1.0. Approximately 100 cells from each culture were then placed on YEP-Gal (2% gal per volume) or YEP-Gal+IAA (1mM) and YPD in triplicate and incubated at 30 O C until visible colonies formed. Viability was calculated by dividing the number of colonies on YEP-Gal or YEP-Gal+IAA plates by the number of colonies on YPD plates. Live cell microscopy and mean squared displacement analysis Live cell microscopy was performed using a spinning disc confocal microscope using Nikon Elements AR software on a Nikon Ni-E upright microscope equipped with a 100× (NA, 1.45) oil immersion objectives, a Yokogawa CSU-W1 spinning-disk head, and an Andor iXon 897U EMCCD camera. Fluorophores were excited at 488nm (GFP) and 561nm (mCherry). Time-lapse series were acquired with 15 optical slices of 0.3µm thickness every 30 seconds for 20 minutes. Time-lapse image stacks were analyzed using a custom MATLAB program designed by the Bloom lab as previously described [60]. Coordinates of DSB and the SPB were tracked with Speckle Tracker, a custom MATLAB program [61, 62]. A custom PERL script was used to convert the pixels to nanometers and subtract the distance of the SPB from the Ddc2-GFP coordinates to eliminate cell and nuclear motion, subtract the mean position of the corrected GFP coordinates, calculate the MSD of each time lapse and export MSD coordinates and radius of confinement (R c ) to an Excel spreadsheet [60]. In MATLAB spot positions were fitted to [µ x , σ x ] = normfit (x – x mean ) and [µ y , σ y ] = normfit (y – y mean ). The variance of the distribution of spot position was then calculated as σ 2 = mean (σ x 2 , σ y 2 ). The average squared deviation from the mean position is (∆r 0 2 ) = (∆x 0 2 ) + (∆y 0 2 ). Using σ 2 and (∆r 0 2 ), we calculated R c as Due to variations in day-to-day collection, wildtype data was only compared to data collected on the same day. Statistical analysis Statistical analysis for Rc comparison was done in PRISM using a t-test or one-way ANOVA analysis. For one-way ANOVA analysis, Dunnett test was used to correct for multiple comparisons. Wildtype comparisons were conducted with data collected from the same day. Resection and cutting assays Resection was measured by quantitative PCR (qPCR) analysis using a restriction enzyme digest as previously described [29]. Briefly, cells are grown in YEP-Lac as described above. 50ml of culture was harvested and DNA extracted using a DNA extraction kit (Masterpure Yeast DNA Purification Kit Cat# MPY80200). DNA was diluted to 10ng/µl. Sty1-HF (digest) or an equivalent amount of water (mock) in Cutsmart buffer at 37C O for 4 h. qPCR samples were run in triplicate on a Biorad CFX384 Real-Time System C1000 Touch Thermal Cycler qPCR machine using Bio-Rad CFX Maestro 1.1 Version 4.1.2433.1219. ADH1 was used as a control gene. See Table S4 for primers. Resection was calculated measuring the fraction of cells that had passed the Sty1 restriction site (RS). f is the fraction of cells where HO has been cleaved. E RS and E ADH1 are the primer efficiencies for the primer pairs 0.7kb, 5kb, 10kb away from the HO-cut site and the ADH1 primers. ∆C q (digest–mock) is the difference between quantification cycles between the mock and digested samples. For cutting assays, DNA was collected as described above. DNA was diluted to 10ng/µl and run using primers flanking the HO-cut site in the MAT locus on chromosome III. ADH1 was used as a control. Gal-HO cutting was measured by the fold increase with the following equation using the 0 h timepoint as a control. TCA protein extraction Protein extracts were prepared for western blot analysis by the standard TCA protocol described in [63]. Briefly, 15-10ml of harvested cells were incubated on ice in 1.5ml microcentrifuge tubes with 20% TCA for 20 minutes. Cells were washed with acetone and the pellet was air dried. 200ul of MURBs buffer (50mM sodium phosphate, 25mM MES, 3M urea, 0.5% 2-mercaptoethanol, 1 mM sodium azide, and 1% SDS) was added to each sample allow with acid washed glass beads. Cells were lysed by mechanical shearing with glass beads for 2 minutes. Supernatant was collected by poking a hole in the bottom of the 1.5ml microcentrifuge tube and spun in a 15ml conical tube. Samples were boiled at 95C for 10 minutes. Western blotting Denatured protein samples prepared by TCA extraction were centrifuged at max speed for 1 minute and 8 – 20 µl of samples were loaded into a 10% or 8% SDS page gel. Proteins were separated by applying 90 V constant until the 37 kDa marker reached the bottom of the gel. Gels were then transferred to an Immun-Blot PVDF using a wet transfer apparatus set to 100 V constant voltage for 1 h. The resulting membranes were blocked in 5% nonfat dry milk or OneBlock buffer (Genesee Scientific, 20-313) for 1 h at room temperature or overnight at 4 °C with gentle agitation. After washing 3 times with 1x TBS-T, blots were incubated with either mouse anti-Myc [9E11] (Abcam, ab56) to detect Tir1 and AID fusions, rabbit anti-Rad53 (Abcam, ab104232), or mouse anti-Pgk1 antibody (Abcam Cat# ab113687, RRID:AB_10861977) for 1 h at room temperature. Blots were then washed 3 times with 1x TBS-T and incubated with anti-mouse or anti-rabbit HRP secondary antibody for 1 h at room temperature. After washing 3 times with 1x TBS-T, ECL Prime was added to fully coat the blots and left to incubate for 5 min at room temperature with gentle agitation. Blots were imaged using a BioRad ChemiDoc XR+ imager and prepared for publication using Image Lab software (BioRad) and Adobe Photoshop CC 2017. Declarations Competing Interests The authors declare no competing interests. Acknowledgments We are grateful to Kerry Bloom for training in MSD measurements, Bruce Goode for donation of CK-666 and expertise, Helle Ulrich for donations of the AID and TIR1 plasmids, David Drubin for donations of the Myo5 plasmids and Susan Gasser for donation of strains. Research was supported by NIH grant R35 GM127029. F.Y.Z, M.A., N.A., K.C., and K.B.F. were supported by NIH Genetics Training Grant TM32GM007122. Author Contributions Conceptualization and initial data collection was conducted by Felix Y. Zhou and James E. Haber. Data collection, imaging, primer design, data analysis, and strain making was conducted by Felix Y. Zhou, Marissa Ashton, Yiyang Jiang, Neha Arora, Kevin Clark, and Kate B. Fitzpatrick. The manuscript was authored and edited by Felix Y. Zhou and James E. Haber. LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for reagents may be directed to, and will be fulfilled by the corresponding author, Dr. James E. Haber ( [email protected] ). References Haber JE (2016) A Life Investigating Pathways That Repair Broken Chromosomes. Annu Rev Genet 50:1–28 Dion V et al (2012) Increased mobility of double-strand breaks requires Mec1, Rad9 and the homologous recombination machinery. Nat Cell Biol 14(5):502–509 Mine-Hattab J, Rothstein R (2012) Increased chromosome mobility facilitates homology search during recombination. Nat Cell Biol 14(5):510–517 Seeber A, Hauer MH, Gasser SM (2018) Chromosome Dynamics in Response to DNA Damage. Annu Rev Genet 52:295–319 Haber JE (2018) DNA Repair: The Search for Homology. BioEssays 40(5):e1700229 Caridi CP et al (2018) Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature 559(7712):54–60 Schrank BR et al (2018) Nuclear ARP2/3 drives DNA break clustering for homology-directed repair. Nature 559(7712):61–66 Goode BL, Eskin JA, Wendland B (2015) Actin and endocytosis in budding yeast. Genetics 199(2):315–358 Symons M et al (1996) Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization. Cell 84(5):723–734 Taylor MD et al (2010) Nuclear role of WASp in the pathogenesis of dysregulated TH1 immunity in human Wiskott-Aldrich syndrome. Sci Transl Med 2(37):37ra44 Han SS et al (2022) WASp modulates RPA function on single-stranded DNA in response to replication stress and DNA damage. Nat Commun 13(1):3743 Galletta BJ, Chuang DY, Cooper JA (2008) Distinct roles for Arp2/3 regulators in actin assembly and endocytosis. PLoS Biol 6(1):e1 Waterman DP et al (2019) Live cell monitoring of double strand breaks in S. cerevisiae. PLoS Genet 15(3):e1008001 Zhang Y, Dudko OK (2016) First-Passage Processes in the Genome. Annu Rev Biophys 45:117–134 Lee SE et al (1998) Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94(3):399–409 Lee CS et al (2014) Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break. Nat Struct Mol Biol 21(1):103–109 Li K et al (2020) Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break. Proc Natl Acad Sci U S A 117(35):21354–21363 Moore JK, Haber JE (1996) Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol 16(5):2164–2173 Idrissi FZ et al (2012) Ultrastructural dynamics of proteins involved in endocytic budding. Proc Natl Acad Sci U S A 109(39):E2587–E2594 Cheblal A et al (2020) DNA Damage-Induced Nucleosome Depletion Enhances Homology Search Independently of Local Break Movement. Mol Cell 80(2):311–326e4 Robertson AS et al (2009) The WASP homologue Las17 activates the novel actin-regulatory activity of Ysc84 to promote endocytosis in yeast. Mol Biol Cell 20(6):1618–1628 Morawska M, Ulrich HD (2013) An expanded tool kit for the auxin-inducible degron system in budding yeast. Yeast 30(9):341–351 Nishimura K et al (2009) An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6(12):917–922 Winter D, Lechler T, Li R (1999) Activation of the yeast Arp2/3 complex by Bee1p, a WASP-family protein. Curr Biol 9(9):501–504 Lewellyn EB et al (2015) An Engineered Minimal WASP-Myosin Fusion Protein Reveals Essential Functions for Endocytosis. Dev Cell 35(3):281–294 Evangelista M et al (2000) A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex. J Cell Biol 148(2):353–362 Geli MI, Riezman H (1996) Role of type I myosins in receptor-mediated endocytosis in yeast. Science 272(5261):533–535 Goodson HV et al (1996) Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton. J Cell Biol 133(6):1277–1291 Gnugge R, Oh J, Symington LS (2018) Processing of DNA Double-Strand Breaks in Yeast. Methods Enzymol 600:1–24 Zierhut C, Diffley JF (2008) Break dosage, cell cycle stage and DNA replication influence DNA double strand break response. EMBO J 27(13):1875–1885 Chen H, Lisby M, Symington LS (2013) RPA coordinates DNA end resection and prevents formation of DNA hairpins. Mol Cell 50(4):589–600 Costelloe T et al (2012) The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection. Nature 489(7417):581–584 Eapen VV et al (2012) The Saccharomyces cerevisiae chromatin remodeler Fun30 regulates DNA end resection and checkpoint deactivation. Mol Cell Biol 32(22):4727–4740 Zhu Z et al (2008) Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134(6):981–994 Mimitou EP, Symington LS (2008) Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455(7214):770–774 Lydeard JR et al (2010) Sgs1 and exo1 redundantly inhibit break-induced replication and de novo telomere addition at broken chromosome ends. PLoS Genet 6(5):e1000973 Groelly FJ et al (2023) Targeting DNA damage response pathways in cancer. Nat Rev Cancer 23(2):78–94 Waterman DP, Haber JE, Smolka MB (2020) Checkpoint Responses to DNA Double-Strand Breaks. Annu Rev Biochem 89:103–133 Mantiero D et al (2007) Dual role for Saccharomyces cerevisiae Tel1 in the checkpoint response to double-strand breaks. EMBO Rep 8(4):380–387 Pellicioli A et al (2001) Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest. Mol Cell 7(2):293–300 Limbo O et al (2011) Mre11 nuclease activity and Ctp1 regulate Chk1 activation by Rad3ATR and Tel1ATM checkpoint kinases at double-strand breaks. Mol Cell Biol 31(3):573–583 Sanchez Y et al (1999) Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science 286(5442):1166–1171 Dubrana K et al (2007) The processing of double-strand breaks and binding of single-strand-binding proteins RPA and Rad51 modulate the formation of ATR-kinase foci in yeast. J Cell Sci 120(Pt 23):4209–4220 Zou L, Elledge SJ (2003) Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300(5625):1542–1548 Longhese MP et al (1998) DNA damage checkpoint in budding yeast. EMBO J 17(19):5525–5528 Dotiwala F et al (2010) Mad2 prolongs DNA damage checkpoint arrest caused by a double-strand break via a centromere-dependent mechanism. Curr Biol 20(4):328–332 Usui T, Ogawa H, Petrini JH (2001) A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol Cell, 7(6): pp. 1255-66 Kim JA, Haber JE (2009) Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete. Proc Natl Acad Sci U S A 106(4):1151–1156 Lee CS et al (2016) Chromosome position determines the success of double-strand break repair. Proc Natl Acad Sci U S A 113(2):E146–E154 Vaze MB et al (2002) Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase. Mol Cell 10(2):373–385 Gautreau AM et al (2022) Nucleation, stabilization, and disassembly of branched actin networks. Trends Cell Biol 32(5):421–432 Clerici M et al (2005) The Saccharomyces cerevisiae Sae2 protein promotes resection and bridging of double strand break ends. J Biol Chem 280(46):38631–38638 Clerici M et al (2008) The Yku70-Yku80 complex contributes to regulate double-strand break processing and checkpoint activation during the cell cycle. EMBO Rep 9(8):810–818 Shroff R et al (2004) Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr Biol 14(19):1703–1711 Usui T, Petrini JH (2007) The Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 directly influence the DNA damage-dependent functions of Rad53. Proc Natl Acad Sci U S A 104(8):2797–2802 Anderson BL et al (1998) The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization. J Cell Biol 141(6):1357–1370 Sun Y, Martin AC, Drubin DG (2006) Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity. Dev Cell 11(1):33–46 Wach A et al (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10(13):1793–1808 Gallagher DN et al (2020) A Rad51-independent pathway promotes single-strand template repair in gene editing. PLoS Genet 16(10):e1008689 Lawrimore J et al (2017) Microtubule dynamics drive enhanced chromatin motion and mobilize telomeres in response to DNA damage. Mol Biol Cell 28(12):1701–1711 Wan X et al (2012) The coupling between sister kinetochore directional instability and oscillations in centromere stretch in metaphase PtK1 cells. Mol Biol Cell 23(6):1035–1046 Wan X et al (2009) Protein architecture of the human kinetochore microtubule attachment site. Cell 137(4):672–684 Miller-Fleming L et al (2014) Detection of Saccharomyces cerevisiae Atg13 by western blot. Autophagy 10(3):514–517 Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5125308","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":362036243,"identity":"61982dbc-f771-4aaf-b886-3cd612293897","order_by":0,"name":"Felix Y. 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Haber","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACAwYeIFlxAMJOAFEHiNJyhmQtjG1QLQzEaDFnP3vwceW8O/n8s5s3Pnjwh0GO70YCfi2WPXnJhme3PbOccedYsUFiG4OxJCEtBgdyzCQbtx02YLiRYyaR2MCQuIGglvNvzH82zjlsIH8jx/xHwh+GesJagIYzNjYcNgAxGBLYGBIMCGt5lyzZcOyZgeGNtGKJxDYJw5lnHhByWO7Bjw01dwzkbiRv/Pjjj40833ECtqADCdKUj4JRMApGwSjADgCiEU6Shh9dnAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1878-0610","institution":"Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, MA, USA","correspondingAuthor":true,"prefix":"","firstName":"James","middleName":"E.","lastName":"Haber","suffix":""}],"badges":[],"createdAt":"2024-09-20 18:05:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5125308/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5125308/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-62377-7","type":"published","date":"2025-08-05T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66061378,"identity":"a0db607a-93e1-4b36-94c3-ac6a0c514afc","added_by":"auto","created_at":"2024-10-07 10:16:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibiting Arp2/3 activity lowers DSB mobility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Mean squared displacement (MSD) analysis (∆t=30s) of the DSB relative to the SPB in FZ015 (WT) strain (n=14) and WT strain with CK-666 (10 µM) (n=34) 3 h after galactose induction of an HO endonuclease-induced irreparable DSB at the \u003cem\u003eMAT\u003c/em\u003e locus on chromosome III. Resected DSBs were bound by Ddc2-GFP and the SPBs were labeled with Spc42-mCherry. Cells were imaged with a 488 nm and 561 nm laser taking optical stacks of 1.5 µm with step size of 300 nm every 30 s for 20 min. See Methods for MSD analysis. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Boxplot of the radius of confinement (Rc) from MSD measurements in (A). Statistical analysis for the radius of confinement derived in PRISM using an unpaired t-test (ns p ≥ 0.05, *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001). See \u003cstrong\u003eTables S1\u003c/strong\u003e and \u003cstrong\u003eS2 \u003c/strong\u003efor significance and R\u003csub\u003eC\u003c/sub\u003e values. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Diagram of experimental setup to measure the MSD of a DSB labeled with Ddc2-GFP as described in (A). CK-666 (10 µM) was added 2 h 40 min after adding galactose.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t=30s) of the DSB relative to the SPB in FZ015 (n=10) and FZ077 (\u003cem\u003esla2∆\u003c/em\u003e) (n=20) 3 h after Gal-HO induction. Imaging and analysis done as described in (A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Boxplot of Rc from MSD measurements in (D). Statistical analysis as described in (B). See \u003cstrong\u003eTables S1\u003c/strong\u003e and \u003cstrong\u003eS2 \u003c/strong\u003efor significance and R\u003csub\u003eC\u003c/sub\u003e values. Mean ± SEM is shown.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/11890a15eb0f2caf30096bb2.png"},{"id":66061380,"identity":"d9dea76e-47c2-4e94-a493-ec68a35feac9","added_by":"auto","created_at":"2024-10-07 10:16:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":142090,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot of the Rc from MSD measurements in (B), (D), and (F). Statistical analysis as described in (B). See \u003cstrong\u003eTable S1 \u003c/strong\u003eand \u003cstrong\u003eS2\u003c/strong\u003e for significance and R\u003csub\u003eC\u003c/sub\u003e values. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eArp2/3 is required for DSB-induced chromatin mobility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Sketch of the GFP-tagged chromosome locus in FZ152. \u003cem\u003eLacO\u003c/em\u003e-array is 4.4kb away from a Gal-HO inducible DSB in the MAT locus on chromosome III labeled with GFP-LacI. SPB is labeled with Spc42-mCherry.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t=30s) of the \u003cem\u003eMAT\u003c/em\u003e locus relative to the SPB before Gal-HO induction (uncut, 0h) (n=11), treatment with CK-666 (10µM) before Gal-HO induction (uncut + CK-666, 0h) (n=10), and 3 h after Gal-HO induction (cut, 3h) (n=16). Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Diagram of experimental setup to measure the MSD of a GFP-LacI/\u003cem\u003elacO \u003c/em\u003earray 4.4 kb away from the HO cut-site on chromosome III. CK-666 was added 20 minutes before adding galactose and MSD was measured 3 h after DSB induction.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t=30s) of the \u003cem\u003eMAT\u003c/em\u003e locus relative to the SPB before Gal-HO induction (uncut, 0h) (n=11), 3 h after Gal-HO induction with CK-666 (10 µM) (cut + CK-666 after gal, 3h) (n=17), and 3 h after Gal-HO induction (cut, 3h) (n=16). Cells treated with CK-666 were given CK-666 20 minutes before imaging. Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Diagram of experimental setup to measure the MSD of a GFP-LacI/\u003cem\u003elacO \u003c/em\u003earray 4.4 kb away from the HO cut-site on chromosome III. CK-666 was added 2 h 40 min after galactose and MSD was measured 3 h after DSB induction.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t=30s) of the \u003cem\u003eMAT\u003c/em\u003e locus relative to the SPB before Gal-HO induction (uncut, 0h) (n=11), 3 h after Gal-HO induction where CK-666 (10 µM) was added 20 minutes before galactose (cut + CK-666 before gal, 3h) (n=10), and 3 h after Gal-HO induction (cut, 3h) (n=16). Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/2506ec3d1573ddea4fe657b8.png"},{"id":66061384,"identity":"ac430f5a-907c-4956-9470-6a86b15d4084","added_by":"auto","created_at":"2024-10-07 10:16:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109908,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot of the Rc from MSD measurements in (A) and (B). Statistical analysis as in 1B. See \u003cstrong\u003eTables S1 \u003c/strong\u003eand \u003cstrong\u003eS2\u003c/strong\u003e for significance and R\u003csub\u003eC\u003c/sub\u003e values. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDegradation of Las17 lowers DSB mobility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Model of the functional domains of Las17, Vrp1, Myo3, and Myo5. Arrows indicate know interactions between these proteins. Not to scale.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Western blot analysis of Las17-AID ± auxin (IAA) (1 mM). IAA or an equivalent amount of 200 proof ethanol was added 2 h after adding galactose. Mouse α-Myc probed for Las17-AID.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t=30s) of DSB relative to the SPB in FZ015 (n=11), FZ075 (Las17-AID) (n=12), and Las17-AID with IAA (1 mM) (Las17-AID + IAA) (n=12) 3 h after Gal-HO induction. Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Boxplot of the Rc from MSD measurements in (C). Statistical analysis as in 1B. See \u003cstrong\u003eTables S1\u003c/strong\u003e and \u003cstrong\u003eS2\u003c/strong\u003e for significance and R\u003csub\u003eC\u003c/sub\u003e values. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t=30s) of DSB relative to the SPB in wildtype (n=13), FZ099 (\u003cem\u003elas17-CA∆\u003c/em\u003e) (n=12), and FZ100 (\u003cem\u003elas17-WH2-CA∆\u003c/em\u003e) (n=15) 3 h after Gal-HO induction. Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/7e7b81a9fd67a7d5a0b26e93.png"},{"id":66061379,"identity":"85eb12e2-1fbf-4d0b-a547-c3a1c878e1da","added_by":"auto","created_at":"2024-10-07 10:16:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe role of Myo3 and Myo5 in DSB mobility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t=30s) of DSB relative to the SPB in FZ015 (WT) (n=21), FZ033 (\u003cem\u003emyo3∆\u003c/em\u003e) (n=16), and FZ034 (\u003cem\u003emyo5∆\u003c/em\u003e) (n= 13) 3 h after Gal-HO induction. Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t=30s) of the DSB in FZ015 (WT) (n=21), FZ033 (\u003cem\u003emyo3∆\u003c/em\u003e) (n=16), and FZ047 (2 copies of Myo5 \u003cem\u003emyo3∆\u003c/em\u003e) (n= 13) 3 h after Gal-HO induction. Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Boxplot of the Rc from MSD measurements in (A) and (B). Statistical analysis as in 1B. See \u003cstrong\u003eTables S1 \u003c/strong\u003eand \u003cstrong\u003eS2\u003c/strong\u003e for significance and R\u003csub\u003eC\u003c/sub\u003e values. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t=30s) of DSB relative to the SPB in FZ015 (WT) (n=24), FZ074 (\u003cem\u003emyo3∆ \u003c/em\u003eMyo5-AID) (n=22), and FZ074 (\u003cem\u003emyo3∆ \u003c/em\u003eMyo5-AID) + IAA (1mM) (n= 19) 3 h after Gal-HO induction. Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Boxplot of the Rc from MSD measurements in (D). Statistical analysis as in 1B. See \u003cstrong\u003eTables S1 \u003c/strong\u003eand \u003cstrong\u003eS2\u003c/strong\u003e for significance and R\u003csub\u003eC\u003c/sub\u003e values. Mean ± SEM is shown.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/19e4ee6a76b9aa294810bb8f.png"},{"id":66061382,"identity":"5ccc9e94-3e3b-4403-84db-74e552357f30","added_by":"auto","created_at":"2024-10-07 10:16:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArp2/3 is required to initiate resection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Percentage of cells with a GFP-LacI/\u003cem\u003elacO\u003c/em\u003e focus in FZ152. CK-666 (10 µM) was added either 1 h before galactose (black) or 1 h after adding galactose (red). Untreated cells are shown in green. Samples were collected every hour after adding galactose for up to 4 h. Cells were imaged with a 488 nm laser taking optical stacks of 1.5 µm with step size of 300 nm. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eB. qPCR-based resection assay. Pairs of primers are placed 10 kb, 5 kb, and 0.7 kb upstream of the HO-cut site. Between each set of primers is a \u003cem\u003eSty\u003c/em\u003eI cleavage site. \u003cem\u003eSty\u003c/em\u003eI is unable to cut ssDNA. See \u003cstrong\u003eMethods\u003c/strong\u003e for more details.\u003c/p\u003e\n\u003cp\u003eC. Resection analysis of FZ015 (WT) and FZ075 (Las17-AID). IAA was added either 1 h before galactose or 1 h after galactose. Samples were collected at 0, 1, 3, 6 h after adding galactose.\u003c/p\u003e\n\u003cp\u003eD. Resection analysis of FZ015 (WT), FZ033 (\u003cem\u003emyo3∆\u003c/em\u003e), and FZ034 (\u003cem\u003emyo5∆\u003c/em\u003e). IAA was added either 1 h before galactose or 2 h after galactose. Samples were collected at 0, 1, 3, 6 h after adding galactose.\u003c/p\u003e\n\u003cp\u003eResection analysis of FZ015 (WT) and FZ074 (\u003cem\u003emyo3∆ \u003c/em\u003eMyo5-AID). IAA was added either 1 h before galactose or 1 h after galactose. Samples were collected at 0, 1, 2, 3, 4, 5, 6 h after adding galactose.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/cf7114bb89d0965e7835f973.png"},{"id":66061387,"identity":"17e95222-bbc4-4ba5-a8c3-2aa5125df8c1","added_by":"auto","created_at":"2024-10-07 10:16:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in the rate of resection lower DSB mobility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Resection analysis of FZ015 (WT) and FZ151 (\u003cem\u003efun30∆\u003c/em\u003e). IAA was added either 1 h before galactose or 1 h after galactose. Samples were collected at 0, 1, 3, 6 h after adding galactose.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t = 30s) of DSB relative to the SPB in FZ015 (WT) (n=17) and FZ151 (\u003cem\u003efun30∆\u003c/em\u003e) (n=12) 3 h after Gal-HO induction. Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Boxplot of the R\u003csub\u003eC\u003c/sub\u003e from MSD measurements in (B). Statistical analysis as in 1B. See \u003cstrong\u003eTables S1\u003c/strong\u003e and\u003cstrong\u003e \u003c/strong\u003eS\u003cstrong\u003e2\u003c/strong\u003e for significance and R\u003csub\u003eC\u003c/sub\u003e values. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t = 30s) of DSB relative to the SPB in FZ015 (WT) (n=23), FZ159 (\u003cem\u003eexo1∆\u003c/em\u003e DNA2-AID) (n=11), and FZ159 (\u003cem\u003eexo1∆\u003c/em\u003e DNA2-AID) + IAA (n=12) 3 h after Gal-HO induction. IAA (1 mM) was added 1 h before image collection. Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Boxplot of the R\u003csub\u003eC\u003c/sub\u003e from MSD measurements in (D). Statistical analysis as in 1B. See \u003cstrong\u003eTables S1\u003c/strong\u003e and\u003cstrong\u003e S2\u003c/strong\u003e for significance and R\u003csub\u003eC\u003c/sub\u003e values. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t = 30s) of DSB relative to the SPB in FZ015 (n=13), FZ015 with CK-666 (10µM) (n=10), and FZ201 (\u003cem\u003epGal:exo1\u003c/em\u003e) (n=14) 3 h after Gal-HO induction. CK-666 (10 µM) was added 20 min before image collection as described in (1A). Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eG.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; MSD analysis (∆t = 30s) of DSB relative to the SPB in FZ015 (n=13), FZ015 with CK-666 (10µM) (n=10), and FZ201 with CK-666 (10µM) (\u003cem\u003epGal:exo1\u003c/em\u003e) (n=13) 3 h after Gal-HO induction. CK-666 (10 µM) was added 20 min before image collection as described in (1A). Imaging and analysis done as described in (1A). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eH.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Boxplot of the R\u003csub\u003eC\u003c/sub\u003e from MSD measurements in (F) and (G). Statistical analysis as in 1B. See \u003cstrong\u003eTables S1\u003c/strong\u003e and\u003cstrong\u003e S2\u003c/strong\u003e for significance and R\u003csub\u003eC\u003c/sub\u003e values. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eI.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Resection analysis of FZ201 (\u003cem\u003epGal:exo1\u003c/em\u003e). Samples were collected at 0, 1, 2, 3, 4, 5, 6 h after adding galactose.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/1d53bd31cc7278856a295da5.png"},{"id":66062297,"identity":"d04642d8-a900-47a4-bb22-2b27ca07759b","added_by":"auto","created_at":"2024-10-07 10:24:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":129958,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Tel1-Mre11 (TM) checkpoint prolongs checkpoint arrest when Arp2/3 is blocked before DSB induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Diagram of experimental setup to measure the percentage of G\u003csub\u003e2\u003c/sub\u003e/M arrested cells after DSB induction. CK-666 or IAA were added 20 minutes or 1 h before galactose to block Arp2/3 activity before DSB induction. To block Arp2/3 activity after DSB induction, CK-666 or IAA were added 2 h after galactose.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Above, percentage of G\u003csub\u003e2\u003c/sub\u003e/M arrested cells in a FZ015 (WT), FZ019 (\u003cem\u003emre11∆\u003c/em\u003e) ± CK-666, and YSL56 (\u003cem\u003etel1∆\u003c/em\u003e)\u003cem\u003e \u003c/em\u003e± CK-666 strains where CK-666 was added before galactose as described in (A). Data are shown from 3 independent experiments with error bars representing standard error of the mean (SEM). Below, western blot of WT and \u003cem\u003emre11∆\u003c/em\u003e strains after DSB induction probed with α-Rad53 shows both an unphosphorylated and phosphorylated protein and α-Pgk1 as a loading control.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Percentage of G\u003csub\u003e2\u003c/sub\u003e/M arrested cells in FZ015 (WT) ± CK-666, FZ033 (\u003cem\u003emyo3∆\u003c/em\u003e), and FZ034 (\u003cem\u003emyo5∆\u003c/em\u003e) after DSB induction. CK-666 was added before galactose as described in (A). Western blot of \u003cem\u003emyo5∆ \u003c/em\u003eprobed with α-Rad53 shows both an unphosphorylated and phosphorylated protein and α-Pgk1 as a loading control.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Percentage of G\u003csub\u003e2\u003c/sub\u003e/M arrested cells in FZ015 (WT) and FZ075 (Las17-AID) ± IAA after DSB induction. IAA was added before galactose as described in (A). Western blot of Las17-AID ± IAA\u003cem\u003e \u003c/em\u003eprobed with α-Myc to show Las17-AID degradation, α-Rad53 shows both an unphosphorylated and phosphorylated protein, and α-Pgk1 as a loading control.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Percentage of G\u003csub\u003e2\u003c/sub\u003e/M arrested cells in FZ015 (WT) and FZ192 (Las17-AID \u003cem\u003emre11∆\u003c/em\u003e) ± IAA DSB induction. IAA was added before galactose as described in (A). Western blot of Las17-AID \u003cem\u003emre11∆\u003c/em\u003e ± IAA\u003cem\u003e \u003c/em\u003eprobed with α-Myc to show Las17-AID degradation, α-Rad53 shows both an unphosphorylated and phosphorylated protein, and α-Pgk1 as a loading control.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Percentage of G\u003csub\u003e2\u003c/sub\u003e/M arrested cells in FZ015 (WT) ± CK-666 and FZ075 (Las17-AID) ± IAA after DSB induction. CK-666 or IAA were added 2 h after galactose as described in (A). Western blot of Las17-AID ± IAA\u003cem\u003e \u003c/em\u003eprobed with α-Myc to show Las17-AID degradation, α-Rad53 shows both an unphosphorylated and phosphorylated protein, and α-Pgk1 as a loading control.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/43ca30be0686ce4b639376c1.png"},{"id":66061388,"identity":"decf7087-6ceb-4a5d-aecd-5e2cbe528f09","added_by":"auto","created_at":"2024-10-07 10:16:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":71699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of NPFs on DSB repair by gene conversion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Model of repair strain YJK17. The donor\u003cem\u003e MAT\u003c/em\u003e\u003cstrong\u003ea\u003c/strong\u003e-inc on chromosome 5 is used as a donor to repair the Gal-HO cut in the MAT locus on chromosome 3 through gene conversion (GC).\u003c/p\u003e\n\u003cp\u003eB. Survival assay in YJK17 mutants. ~200-100 cells from a YP-Lac culture were plated on YPD, YP-Gal, and YP-Gal+IAA plates. The survival rate was calculated by the number of cells grown on a YEP-Gal or YEP-Gal+IAA plate divided by the number of cells grown on a YPD plate. See \u003cstrong\u003eMethods \u003c/strong\u003efor more details. Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eC. Model of repair strains with an HO cut site inserted at \u003cem\u003eLEU2 \u003c/em\u003eon chromosome 5 and a \u003cem\u003eLEU2 \u003c/em\u003edonor at different locations.\u003c/p\u003e\n\u003cp\u003eD. Survival assay comparing survival rates of wildtype strains with their \u003cem\u003emyo5∆\u003c/em\u003ecounterparts. Survival assay conducted as described in (B). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eE. Survival assay comparing survival rates of wildtype strains with their \u003cem\u003eLAS17-AID\u003c/em\u003ecounterparts ± IAA (1mM). Survival assay conducted as described in (B). Mean ± SEM is shown.\u003c/p\u003e\n\u003cp\u003eF. Model of repair strain YMV2 and repair by single-strand annealing (SSA) or break induced replication (BIR). An HO cut-site is inserted in LEU2 on chromosome 3 (\u003cem\u003eLEU2::HOcs\u003c/em\u003e) and a 1.3 kb donor of the C-terminal end of LEU2 (\u003cem\u003eU2\u003c/em\u003e) is inserted 30 kb upstream of \u003cem\u003eLEU2::HOcs\u003c/em\u003e. The 30 kb sequence between the homologous sequences is deleted after repair by either SSA or BIR.\u003c/p\u003e\n\u003cp\u003eG. Survival assay of YMV2 strains grown on YP-Gal-IAA plates. Survival assay conducted as described in (B). Mean ± SEM is shown.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/045a9023d382fefda8264021.png"},{"id":88402471,"identity":"a83f7806-929b-44b7-9c60-ec733a78c10f","added_by":"auto","created_at":"2025-08-06 07:10:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2284025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/66dd6d87-75e6-4a45-83f4-3b109b47b313.pdf"},{"id":66061386,"identity":"f9150253-e89e-41a8-b065-2e3e7a486fbe","added_by":"auto","created_at":"2024-10-07 10:16:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":790254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"DSBMobilityManuscriptNatureCommunicationsSupplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/869622d8b8d7b60f25c7b3ed.docx"},{"id":66061381,"identity":"bcd5f3e1-3949-4429-acdb-a57a9cc80bb6","added_by":"auto","created_at":"2024-10-07 10:16:22","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2394965,"visible":true,"origin":"","legend":"\u003cp\u003eReporting Summary\u003c/p\u003e","description":"","filename":"reportingsummary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5125308/v1/0331d328f81510932bdfed71.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Arp2/3 and Type-I myosins control chromosome mobility and end-resection at double-strand breaks in S. cerevisiae","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRepair of double-strand breaks (DSBs) is required to maintain genome integrity. Homologous recombination (HR) requires that the ends of the DSB find a homologous donor sequence to use as a template for repair (reviewed in [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]). In budding yeast, a DSB causes an increase in chromatin mobility near a DSB break site [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which is thought to facilitate the Rad51-mediated search for homology by increasing the volume that the DSB ends explore within the nucleus, defined as the radius of confinement (Rc) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While processes such as the activation of the Mec1\u003csup\u003eATR\u003c/sup\u003e-dependent DNA damage checkpoint, the phosphorylation of histone H2A, and recruitment of repair proteins to the break have been shown to be required for DSB-induced chromatin mobility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], there is still no mechanism that explains how a DSB causes increased chromatin mobility or how changes in mobility affect DSB repair (reviewed in [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003eSeveral studies have suggested that actin branching is directly involved in repairing double-strand breaks. Recently it has been shown in mammalian cells and \u003cem\u003eDrosophila\u003c/em\u003e that inactivation of the actin nucleator Arp2/3 by the drug CK-666 lowers DSB mobility [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Treatment of \u003cem\u003eDrosophila\u003c/em\u003e cells with CK-666 or RNAi treatment of nuclear myosin proteins impaired the directed movement of damaged DNA in heterochromatin to the nuclear periphery [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition, CK-666 caused an apparent reduction in the amount of single-stranded DNA (ssDNA) formed at DSB ends [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A proteomics study in \u003cem\u003eXenopus\u003c/em\u003e egg extracts has shown that all seven-subunits of Arp2/3 and actin are enriched at DSBs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe branching of polymerized actin is dependent on the Arp2/3 complex and a set of nucleation promoting factors (NPF) (reviewed by [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The Arp2/3 actin nucleator Wiskott-Aldrich syndrome protein (WASP) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] has been shown to affect the loading of the single-strand binding protein RPA onto single-stranded DNA in mammalian cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and mutations in Las17, the budding yeast homologue for WASP, was shown to have hypersensitivity to the DNA damaging agents hydroxyurea (HU) and methyl methanesulfonate (MMS) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Las17 mutants accumulated more Rad52 foci when exposed to HU or MMS, suggesting a deficiency in repair by HR [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere we have investigated the role of Arp2/3 and its associated NPFs in \u003cem\u003eS. cerevisiae\u003c/em\u003e in more detail. In budding yeast, Arp2/3 and the NPFs Las17 and the type-I myosins (Myo3 and Myo5) (reviewed in [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]) are primarily found in cortical actin patches and are required for clathrin-mediated endocytosis (CME) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These factors are well-studied in endocytosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], but their roles in other cellular processes remain poorly defined.\u003c/p\u003e \u003cp\u003eHere we have used Ddc2-GFP to directly label DSBs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and mean-square displacement (MSD) analysis (reviewed in [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]) to track and measure changes in mobility of an irreparable site-specific DSB at the \u003cem\u003eMAT\u003c/em\u003e locus on chromosome III. We found that blocking Arp2/3 activity, either through the drug CK-666 or through a deletion/mutation of NPFs, lowered DSB mobility. Moreover, 5\u0026rsquo; to 3\u0026rsquo; resection was reduced when Arp2/3 was inactivated after DSB formation. When Arp2/3 activity is blocked before DSB induction, the initiation of resection was severely impaired. Additionally, we show that altering the rate of resection, through deleting the chromatin remodeler Fun30 or inactivating the Exo1 and Dna2 nucleases responsible for long-range resection, lowered DSB mobility. Altogether our study shows that Arp2/3 has an evolutionarily conserved role in regulating both DSB mobility and DSB end-resection, and that directly impairing the rate of resection prevents the increased motion of DSB ends.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLabeling and tracking the mobility of double-stranded breaks\u003c/h2\u003e \u003cp\u003eTo generate, track, and quantify the mobility of a single-irreparable DSB we modified the well-characterized strain JKM179, in which a single site-specific cut in the \u003cem\u003eMAT\u003c/em\u003e locus on chromosome III is induced by a galactose-regulated HO-endonuclease gene (Gal-HO) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To visualize and track the DSB, we monitored the DNA damage checkpoint protein Ddc2 (Ddc2-GFP) which we have previously shown forms a damage-dependent GFP focus in ~\u0026thinsp;80% of cells 3 h after adding galactose [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. An mCherry-tagged spindle pole body protein Spc42 (Spc42-mCherry) was used as a fiducial marker to account for random nuclear motion. Cutting by Gal-HO is highly efficient with nearly 90% of cells being cleaved 1 h after galactose addition [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In these strains the homologous \u003cem\u003eHML\u003c/em\u003e and \u003cem\u003eHMR\u003c/em\u003e donors are deleted, thus preventing repair of \u003cem\u003eMAT\u003c/em\u003e by HR. When HO is continually expressed, repair of the DSB by altering the cleavage site via nonhomologous end-joining occurs in only 0.2% of cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We measured DSB mobility 3 h after adding galactose and quantified the mobility of the DSB using mean-squared displacement (MSD) analysis (see \u003cb\u003eMethods\u003c/b\u003e for details) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBlocking Arp2/3 activity lowers DSB mobility\u003c/h3\u003e\n\u003cp\u003eTo test whether Arp2/3 activity has a conserved role in DSB mobility in budding yeast, we added 10\u0026micro;M CK-666 to block Arp2/3 activity 20 min before imaging (160 min after adding galactose) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Addition of CK-666 significantly lowered the radius of confinement (Rc) of the DSB from 0.92 \u0026micro;m to 0.69 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB \u003cb\u003eand Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Addition of CK-666 did not affect the mobility of the spindle pole bodies relative to the bud neck (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), suggesting that the changes seen in DSB mobility when cells are treated with CK-666 were due to the changes in the behavior of the DSB.\u003c/p\u003e \u003cp\u003eSince Arp2/3 is primarily known as a component of endocytosis in budding yeast, we asked whether the change in DSB mobility was due to impaired endocytosis. Deletion of \u003cem\u003eSLA2\u003c/em\u003e, a key component of endocytosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], did not affect the behavior of the DSB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E \u003cb\u003eand Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). This result suggests that endocytosis is not required for DSB mobility and that the role of Arp2/3 in DSB mobility is independent of its role in endocytosis.\u003c/p\u003e\n\u003ch3\u003eArp2/3 affects changes in local and global chromatin mobility in response to a DSB\u003c/h3\u003e\n\u003cp\u003eWhile the direct labeling of DSBs with Ddc2-GFP is suitable for tracking the mobility of DSBs, it cannot be used to establish a basal level of chromatin mobility in the absence of a DSB. To test whether Arp2/3 is required for increasing chromatin mobility near a DSB, we induced an irreparable DSB at \u003cem\u003eMAT\u003c/em\u003e in a derivative of strain JKM179 that expresses the GFP-LacI protein that binds to a \u003cem\u003elacO\u003c/em\u003e array inserted 4.4 kb away from the \u003cem\u003eMAT\u003c/em\u003e locus [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). As previously reported [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we found that inducing a DSB caused an increase in local chromatin mobility near the DSB site: R\u003csub\u003eC\u003c/sub\u003e 0.63 \u0026micro;m (uncut) and 1.1 \u0026micro;m (cut) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, G \u003cb\u003eand Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). CK-666 did not significantly affect basal levels of chromatin mobility: Rc uncut (0.63 \u0026micro;m) and uncut\u0026thinsp;+\u0026thinsp;CK-666 (0.54 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, G \u003cb\u003eand Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e); however, treatment with CK-666 either 20 min before (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) or 160 min after (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) DSB induction resulted in no increase in DSB mobility compared with the uncut condition, monitored at 180 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, F-G \u003cb\u003eand Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Thus, CK-666 impairs DSB-induced chromatin mobility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eNucleation promoting factors are required DSB-induced increase in mobility\u003c/h3\u003e\n\u003cp\u003eIn humans, Arp2/3 requires the NPF WASP to nucleate new branches of actin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]; Las17 is the budding yeast homologue of WASP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To test whether Las17 was required for DSB mobility, we added an auxin-inducible degron (AID) to the C-terminus of Las17 (Las17-AID) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Adding 1mM auxin (IAA) caused degradation of Lad17-AID within 1h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The AID-tag did not affect the MSD curve of Las17-AID, but when IAA was added 2 h after Gal-HO induction, the mobility of the DSB measured at 3 h dropped: Rc WT (0.83 \u0026micro;m), Las17-AID (0.77 \u0026micro;m), and Las17-AID\u0026thinsp;+\u0026thinsp;IAA (0.49 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further characterize the role of Las17, we deleted functional domains that are responsible for Arp2/3 activation. Deletion of the acidic patch (CA) domain (\u003cem\u003elas17-CA∆\u003c/em\u003e), which directly binds to Arp2/3 to activate Arp2/3 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], did not result in a significant change in the mobility of the DSB: Rc WT (0.91 \u0026micro;m) and \u003cem\u003elas17-CA∆\u003c/em\u003e (0.90 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). However, additional deletion of the WH2 domain, which brings monomeric actin to Arp2/3, along with the CA domain (\u003cem\u003elas17-WH2-CA\u003c/em\u003eΔ) lowered the mobility of the DSBs: R\u003csub\u003eC\u003c/sub\u003e \u003cem\u003elas17-WH2-CA\u003c/em\u003eΔ (0.53 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). This suggests that Las17 in its role as an Arp2/3 actin nucleator is required for DSB mobility.\u003c/p\u003e\n\u003ch3\u003eThe type-I myosins Myo3 and Myo5 play a role in DSB mobility\u003c/h3\u003e\n\u003cp\u003eType-I myosins Myo3 and Myo5 promote actin nucleation by Arp2/3 during endocytosis in budding yeast [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and depletion of Myo1a and Myo1b in \u003cem\u003eDrosophilia\u003c/em\u003e increases sensitivity to ionizing radiation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. We asked if Myo3 or Myo5 have a role in DSB mobility in budding yeast. We found that a single deletion of \u003cem\u003eMYO3\u003c/em\u003e or \u003cem\u003eMYO5\u003c/em\u003e was sufficient to lower the mobility of the DSB: Rc WT (0.83 \u0026micro;m), \u003cem\u003emyo3∆\u003c/em\u003e (0.60 \u0026micro;m), and \u003cem\u003emyo5∆\u003c/em\u003e (0.58 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C \u003cb\u003eand Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The effect of deleting either type-I myosin was different from what is seen in endocytosis, where Myo3 and Myo5 are functionally redundant [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We then asked whether a second copy of one type-I myosin, added to a deletion of the other, would restore DSB mobility. We integrated a second copy of \u003cem\u003eMYO5\u003c/em\u003e at \u003cem\u003eURA3\u003c/em\u003e on chromosome V, under control of its own promoter, in a \u003cem\u003emyo3\u003c/em\u003eΔ background and found that there was no significant difference in the Rc between the WT (0.83 \u0026micro;m) and the \u003cem\u003emyo3\u003c/em\u003eΔ Myo5 (+\u0026thinsp;Myo5) (0.80 \u0026micro;m) strains. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine which functional domains of Myo5 are required for DSB mobility, we integrated a series of \u003cem\u003eMYO5\u003c/em\u003e domain deletion plasmids with their endogenous promotors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] into a \u003cem\u003emyo5\u003c/em\u003eD strain. In the presence of Myo3, we found that there was no significant difference in the R\u003csub\u003eC\u003c/sub\u003e between the wildtype strain and a deletion of the Myo5 motor-domain, TH1, or TH2 domains: WT (0.89 \u0026micro;m), \u003cem\u003emyo5-motorΔ\u003c/em\u003e (0.86\u0026micro;m), \u003cem\u003emyo5-TH1Δ\u003c/em\u003e (0.85 \u0026micro;m), and \u003cem\u003emyo5-TH2Δ\u003c/em\u003e (0.96 \u0026micro;m) (\u003cb\u003eFigure S2A-C, E and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). However, a deletion of the SH3 domain (\u003cem\u003emyo5-SH3Δ\u003c/em\u003e) did not restore the mobility of the DSBs back to wildtype levels; R\u003csub\u003eC\u003c/sub\u003e WT (0.89 \u0026micro;m) and \u003cem\u003emyo5-SH3Δ\u003c/em\u003e (0.62 \u0026micro;m) (\u003cb\u003eFigure S2D-E, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eBecause a double deletion of \u003cem\u003eMYO3\u003c/em\u003e and \u003cem\u003eMYO5\u003c/em\u003e is synthetically lethal in most backgrounds [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], we added an AID tag to Myo5 (Myo5-AID) in a \u003cem\u003emyo3\u003c/em\u003eD strain. In a \u003cem\u003eMYO3\u003c/em\u003e strain, the addition of the AID tag to Myo5 did not affect the mobility of the DSB and adding IAA 2 h after DSB induction reduced mobility, similar to the \u003cem\u003eMYO5\u003c/em\u003e deletion. (\u003cb\u003eFigure S3, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Unexpectedly, in the \u003cem\u003emyo3∆\u003c/em\u003e Myo5-AID strain, treatment with IAA increased the mobility of the DSB to near WT levels: Rc WT (0.81 \u0026micro;m), \u003cem\u003emyo3∆\u003c/em\u003e Myo5-AID (0.6 \u0026micro;m), and \u003cem\u003emyo3∆\u003c/em\u003e Myo5-AID\u0026thinsp;+\u0026thinsp;IAA (0. 81 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eSince Las17 has a WH2 domain and acidic (CA) patch, it is possible that the increase in DSB mobility when both Myo3 and Myo5 are absent could be due to the Arp2/3 actin nucleation by Las17. To test this, we deleted the WH2 and CA domains of Las17 (\u003cem\u003elas17-WH2-CA\u003c/em\u003eΔ) in a \u003cem\u003emyo3\u003c/em\u003eΔ Myo5-AID background and measured the mobility of the DSB\u0026thinsp;\u0026plusmn;\u0026thinsp;IAA as described above. We found that the \u003cem\u003elas17\u003c/em\u003e mutant prevented the restoration of Rc to WT levels that was seen when both Myo3 and Myo5 are inactivated: Rc WT (0.99 \u0026micro;m), \u003cem\u003emyo3\u003c/em\u003eΔ Myo5-AID \u003cem\u003elas17-WH2-CA\u003c/em\u003eΔ (0.57 \u0026micro;m), and \u003cem\u003emyo3\u003c/em\u003eΔ Myo5-AID \u003cem\u003elas17-WH2-CA\u003c/em\u003eΔ\u0026thinsp;+\u0026thinsp;IAA (0.58 \u0026micro;m) (\u003cb\u003eFigure S4, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Together these results suggest that Myo3 and Myo5 in their role as Arp2/3 actin nucleators play a role in DSB mobility and Las17 can compensate for the loss of both Myo3 and Myo5 to maintain near WT levels of DSB mobility.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eArp2/3 and myosins are required for damage-dependent focus formation with Ddc2-GFP or Rad51-GFP\u003c/h2\u003e \u003cp\u003eDdc2-GFP and Rad51-GFP form damage-dependent foci in \u0026ge;\u0026thinsp;80% of cells 3 h after DSB induction [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, when Arp2/3 activity was blocked by CK-666 before DSB induction, we observed that Ddc2-GFP and Rad51-GFP formed foci in fewer than 15% of cells (\u003cb\u003eFigure S5A-B\u003c/b\u003e). Similar inhibition of the DSB-induced foci was found in a deletion of \u003cem\u003eVRP1\u003c/em\u003e, or when IAA was added to Las17-AID or \u003cem\u003emyo3Δ\u003c/em\u003e Myo5-AID cultures. Deletion of \u003cem\u003eKU70\u003c/em\u003e did not restore damage-dependent Ddc2-GFP foci formation in CK-666 treated cells (\u003cb\u003eFigure S5C\u003c/b\u003e). This finding raised the possibility that blocking Arp2/3 might interfere with either Gal-HO cutting or with long-range resection. By using a pair of PCR primers that flank the HO-cut site in the \u003cem\u003eMAT\u003c/em\u003e locus (\u003cb\u003eFigure S5A\u003c/b\u003e) we showed that Gal-HO cutting was unaffected in Las17-AID or \u003cem\u003emyo3∆\u003c/em\u003e Myo5-AID mutants treated 1 h before DSB induction with IAA (\u003cb\u003eFigure S5B-D\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eArp2/3 and type-I myosins are required for the initiation and maintenance of resection\u003c/h3\u003e\n\u003cp\u003eSince Gal-HO cutting is normal, blocking Arp2/3 activity might interfere with 5\u0026rsquo; to 3\u0026rsquo; resection of the DSB. We first monitored the disappearance of GFP-LacI/\u003cem\u003elacO\u003c/em\u003e foci in a strain where the \u003cem\u003elacO\u003c/em\u003e array was inserted 4.4 kb away from the HO cleavage site at \u003cem\u003eMAT\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). As resection erodes the \u003cem\u003elacO\u003c/em\u003e array, GFP-LacI will lose its binding sites and the GFP-LacI/\u003cem\u003elacO\u003c/em\u003e focus will vanish. Within 2 h after DSB induction there was a steady drop in the percentage of cells with a GFP-LacI focus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, addition of CK-666 1 h before HO induction prevented the loss of the GFP-LacI focus. Resection in cells treated with CK-666 after 1 h after DSB induction was also significantly impaired.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe used a restriction enzyme-based qPCR resection assay to measure the generation of ssDNA around the HO-cut site itself [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. qPCR analysis was done using a series of primers flanking \u003cem\u003eSTY1\u003c/em\u003e cut sites at different distances from the HO-cut site in the \u003cem\u003eMAT\u003c/em\u003e locus (0.7 kb, 5 kb, and 10 kb) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). As resection converts dsDNA to ssDNA, \u003cem\u003eSty\u003c/em\u003eI cleavage sites are lost and qPCR threshold signal is detected.\u003c/p\u003e \u003cp\u003eWe measured resection in Las17-AID, \u003cem\u003emyo3∆\u003c/em\u003e, \u003cem\u003emyo5∆\u003c/em\u003e, and \u003cem\u003emyo3Δ\u003c/em\u003e Myo5-AID mutants. IAA was added either 1 h before or 2 h after adding galactose. Las17-AID without IAA had a similar resection profile as the wildtype; however, when IAA was added either before or after galactose, long range resection was severely impaired (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) suggesting that Las17 is required for the initiation and maintenance of resection.\u003c/p\u003e \u003cp\u003eSingle deletions of \u003cem\u003eMYO3\u003c/em\u003e or \u003cem\u003eMYO5\u003c/em\u003e did not greatly affect resection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Resection in the \u003cem\u003emyo3∆\u003c/em\u003e Myo5-AID mutant without IAA was comparable to resection in the WT, as expected from the \u003cem\u003emyo3∆\u003c/em\u003e resection data (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E); but when IAA was added to \u003cem\u003emyo3∆\u003c/em\u003e Myo5-AID 1 h before DSB induction, there was little to no resection up to 6 h after DSB induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Finally, when IAA was added 2 h after DSB induction, resection in \u003cem\u003emyo3\u003c/em\u003eD Myo5-AID appeared to be greatly inhibited within an hour of degrading Myo5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Together these data suggests that Arp2/3 and type-I myosins have a role both in the initiation of resection and in its maintenance.\u003c/p\u003e\n\u003ch3\u003eChanges in resection lower the mobility of DSBs\u003c/h3\u003e\n\u003cp\u003eSince resection was affected by changes in Arp2/3 activity, we asked whether there was a correlation between the rate of resection and DSB mobility. Previously we and others have shown that a deletion of the chromatin remodeler \u003cem\u003eFUN30\u003c/em\u003e greatly reduced Exo1 and Sgs1/Dna2-dependent long-range resection [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although resection through the 0.7 kb site was not affected in \u003cem\u003efun30∆\u003c/em\u003e, long-range resection past the 5 and 10 kb sites was markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). MSD analysis of a DSB showed the R\u003csub\u003eC\u003c/sub\u003e of \u003cem\u003efun30\u003c/em\u003eD (0.5 \u0026micro;m) was significantly lower than the WT (0.8 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLong-range resection requires both the exonuclease Exo1 and the helicase/endonuclease DNA2-Top3-Rmi1-Sgs1 complex [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Since the rate of resection is not affected by deleting either \u003cem\u003eEXO1\u003c/em\u003e or \u003cem\u003eSGS1\u003c/em\u003e alone [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], we blocked long range resection by deleting \u003cem\u003eEXO1\u003c/em\u003e and degrading Dna2-AID. There was no difference in DSB mobility between the wildtype and \u003cem\u003eexo1\u003c/em\u003eD Dna2-AID, but when IAA was added 2 h after DSB induction the mobility of the DSB measured at 3 h was reduced: Rc WT (0.8 \u0026micro;m), \u003cem\u003eexo1\u003c/em\u003eΔ DNA2-AID (0.8 \u0026micro;m), and \u003cem\u003eexo1\u003c/em\u003eΔ DNA2-AID\u0026thinsp;+\u0026thinsp;IAA (0.5 \u0026micro;m). (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo test whether increasing resection affected DSB mobility, we measured mobility in a strain with a second copy of \u003cem\u003eEXO1\u003c/em\u003e expressed under control of a galactose promoter (pGal:\u003cem\u003eEXO1\u003c/em\u003e) at the \u003cem\u003eLEU2\u003c/em\u003e locus [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We found that there was no difference between the Rc in WT and when Exo1 was overexpressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, H \u003cb\u003eand Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) even though the rate of resection was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). However, pGal::\u003cem\u003eEXO1\u003c/em\u003e suppressed the reduction in Rc from CK-666 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), but did not rescue Ddc2-GFP focus formation in cells treated with CK-666 before DSB induction (\u003cb\u003eFigure S5D\u003c/b\u003e). Taken together, continuous resection is required for the high mobility of DSBs and overexpression of Exo1 can overcome the reduced rate of resection caused by CK-666 but could not prevent the block to the initiation of resection.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eActivation of the TM checkpoint in response to blocking Arp2/3 activity\u003c/h2\u003e \u003cp\u003eWhen a DSB is detected, the DNA damage checkpoint (DDC) is activated to halt cell cycle progression and allow cells a chance to repair the DSB before proceeding through mitosis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In budding yeast, activation of the DDC is mainly controlled through the effector kinase Mec1\u003csup\u003eATR\u003c/sup\u003e with a minor contribution by the Tel1\u003csup\u003eATM\u003c/sup\u003e kinase [\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Mec1 is recruited to DSBs by its binding partner Ddc2\u003csup\u003eATRIP\u003c/sup\u003e, which binds to RPA loaded on ssDNA [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Arrest by the DDC is typically monitored by cell morphology, as cells shift towards a large-budded G\u003csub\u003e2\u003c/sub\u003e/M state, and by Western blot analysis of phosphorylation of the Mec1/Tel1 target, Rad53 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe hypothesized that preventing resection - by blocking Arp2/3 activity prior to HO induction - should interfere with cell cycle arrest through the DDC. Typically, a cell with a single irreparable DSB will arrest for 12\u0026ndash;15 h before escaping arrest through a process known as adaptation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Here we find that when Arp2/3 activity was blocked before DSB induction, either by drug treatment with CK-666 or degradation of Las17-AID with IAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), G\u003csub\u003e2\u003c/sub\u003e/M arrest was shortened to 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-G). Deletion of either Myo3 or Myo5 did not shorten checkpoint arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile Mec1 is primarily responsible for checkpoint arrest by the DNA damage checkpoint, the Tel1-Mre11 (TM) checkpoint is an alternative checkpoint response in response to a DSB, notably in \u003cem\u003emec1\u003c/em\u003eD cells in which initial resection by the Mre11 complex is impaired [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. We confirmed that the short checkpoint response we observed was attributable to the TM checkpoint, as \u003cem\u003etel1\u003c/em\u003eD and \u003cem\u003emre11\u003c/em\u003eD strains treated with CK-666 before DSB induction did not exhibit a checkpoint response (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). A \u003cem\u003emre11Δ\u003c/em\u003e Las17-AID double mutant treated with auxin also showed little or no G\u003csub\u003e2\u003c/sub\u003e/M arrest and western blot analysis showed that Rad53 was not phosphorylated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH-I).\u003c/p\u003e \u003cp\u003eSince degrading Las17 after DSB induction impaired further resection, we assayed whether Arp2/3 is required to maintain checkpoint arrest by adding CK-666 to our wildtype strain or IAA to Las17-AID 2 h after adding galactose and monitored for changes in cell morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We found that treatment with CK-666 or degrading Las17-AID resulted in cells escaping G\u003csub\u003e2\u003c/sub\u003e/M arrest early despite Rad53 remaining hyperphosphorylated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ-K). These results show that the shortened DSB-induced checkpoint arrest was dependent on the TM checkpoint which was activated when resection was impaired by inhibiting Arp2/3 or its associated factors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDSB Mobility and Repair by Gene Conversion\u003c/h2\u003e \u003cp\u003eIf DSB-induced chromatin mobility facilitates homology search, then lowering the mobility of DSBs might impact the repair efficiency carried out by gene conversion (GC). To study how changes in chromatin mobility affect the efficiency of DSB repair, we modified the well-characterized strain YJK17 which has an ectopic, mutated copy of \u003cem\u003eMAT\u003c/em\u003e (\u003cem\u003eMAT\u003c/em\u003e\u003cb\u003ea-inc\u003c/b\u003e) that cannot be cut by the HO-endonuclease and which serves as a donor to repair the cleavage at \u003cem\u003eMAT\u003c/em\u003eα by interchromosomal gene conversion (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. With this strain we could generate and monitor the repair of a single DSB event.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDeletion of \u003cem\u003eMYO3\u003c/em\u003e, \u003cem\u003eMYO5\u003c/em\u003e, or \u003cem\u003eMRE11\u003c/em\u003e did not impact repair; however Las17-AID showed a significant decrease in viability from YP-Gal (79%) and YP-Gal\u0026thinsp;+\u0026thinsp;IAA (46%) plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). An \u003cem\u003emre11Δ\u003c/em\u003e Las17-AID double mutant showed an even more significant drop in viability (65% on YP-Gal and 10% on YP-Gal\u0026thinsp;+\u0026thinsp;IAA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Las17-AID survivors from the YP-Gal and YP-Gal\u0026thinsp;+\u0026thinsp;IAA plates grew normally on YPD and YP-Gal plates (\u003cb\u003eFigure S7\u003c/b\u003e) as expected.\u003c/p\u003e \u003cp\u003eTo further test the effects of mobility-deficient mutants on GC, we deleted \u003cem\u003eMYO5\u003c/em\u003e and modified Las17 in a series of strains we had previously characterized with repair efficiencies ranging from 46\u0026ndash;9% [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The differences in repair efficiencies reflect the contact probability between the DSB site and the \u003cem\u003eLEU2\u003c/em\u003e donor sequence. As with YJK17, we found that \u003cem\u003emyo5\u003c/em\u003eD did not affect repair efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD) but Las17-AID\u0026thinsp;+\u0026thinsp;IAA showed a significant decrease in the percentage of survivors in all 4 strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eSince degradation of Las17 showed reduced resection, we wanted to know how this would affect repair by single-strand annealing (SSA) using the previously characterized strain YMV2, which has an HO cut-site inserted in \u003cem\u003eLEU2\u003c/em\u003e on chromosome 3 (\u003cem\u003eleu2-cs\u003c/em\u003e) and a 1.3 kb fragment of the 3\u0026rsquo; end of \u003cem\u003eLEU2 (U2)\u003c/em\u003e inserted 30 kb upstream of \u003cem\u003eleu2-cs\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The DSB can be repaired both by Rad51-dependent break-induced replication and by Rad51-independent SSA [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Degradation of Las17 reduced the survival rate from 86\u0026ndash;63% (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). Deletion of \u003cem\u003eRAD51\u003c/em\u003e did not affect the repair efficiency, 85% (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). Yet when Las17 was degraded in the \u003cem\u003erad51Δ\u003c/em\u003e background, repair efficiency dropped to 36% (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). This suggests that Las17 and Arp2/3 have a role in repair by SSA and have much less effect on BIR, where much less resection is required.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhen a cell commits to repair a DSB through HR, there is an increase in chromatin mobility near the DSB [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. While it is thought that increased chromatin mobility near a DSB facilitates homology search, it is unclear what role DSB-induced chromatin mobility has in DSB repair by HR. In this study we investigated the role of the Arp2/3 actin nucleator in DSB mobility and repair by HR.\u003c/p\u003e \u003cp\u003eHere we show that Arp2/3, previously shown to be required for DSB mobility in \u003cem\u003eDrosophila\u003c/em\u003e and mammalian cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], has a conserved role in DSB mobility in budding yeast and that this process is separate from Arp2/3\u0026rsquo;s previously well-characterized function in endocytosis [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. It appears that Arp2/3 is required at 2 different steps during homology-based repair: first, for the initiation, and then for the maintenance of long-range resection and, perhaps consequently, for the increased mobility of the DSB during homology searching.\u003c/p\u003e \u003cp\u003eLong-range resection was not seen when Arp2/3 activity was blocked before DSB-induction, either through treatment with the Arp2/3 inhibitor CK-666, deletion of \u003cem\u003eVRP1\u003c/em\u003e, degradation of Las17\u003csup\u003eWASP\u003c/sup\u003e, or when both type-I myosins, Myo3 and Myo5, were not present. Unlike a \u003cem\u003eSAE2\u003c/em\u003e deletion mutant, which has been reported to have delayed DSB-induced chromatin mobility [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and delayed resection [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], we saw little or no resection up to 6 h after inducing an HO break when Arp2/3 was blocked prior to DSB induction.\u003c/p\u003e \u003cp\u003eIn previous studies, deletion of \u003cem\u003eYKU70/80\u003c/em\u003e allowed Exo1-dependent resection of DSB ends in G1-arrested cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and overexpression of \u003cem\u003eYKU70/80\u003c/em\u003e interfered with Mre11 association with DSB ends and acted as a barrier for resection [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Here we show that a deleting \u003cem\u003eYKU70\u003c/em\u003e did not overcome the CK-666 block to forming Ddc2-GFP foci, suggesting that Arp2/3 does not affect the residence of yKu70/80 at DSB ends.\u003c/p\u003e \u003cp\u003eThe connection between increased chromatin mobility and end-resection is complex. As in mammalian cells, slowing down resection reduces the mobility of DSB ends, though whether the same factors are involved remains unclear. In mammals, inhibition of the Mre11 by mirin reduced mobility [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], while in budding yeast inhibition of the Mre11 complex by deletion of \u003cem\u003eSAE2\u003c/em\u003e only delays resection and the increase in Rc [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Here we found that Rc was reduced by impairing long-range resection, either by deleting the chromatin remodeler \u003cem\u003eFUN30\u003c/em\u003e or by inactivation of both Exo1 and Dna2. Thus, a defect in long-range end-resection is sufficient to impair damage-induced chromatin mobility. We also found that \u003cem\u003eEXO1\u003c/em\u003e overexpression suppressed the reduction in Rc caused by adding CK-666, but overexpression of \u003cem\u003eEXO1\u003c/em\u003e was not able to bypass the block in initiating resection when Arp2/3 was inhibited prior to DSB induction. We were unable to test DSB mobility in a triple deletion mutant of the primary resection proteins because an \u003cem\u003emre11-nd\u003c/em\u003e (nuclease dead) \u003cem\u003eexo1 sgs1Δ\u003c/em\u003e deletion is inviable [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]; but it would appear that inactivating Arp2/3 in an \u003cem\u003emre11\u003c/em\u003eD background eliminates both long- and short-range resection and thus recombination. These findings confirm and extend the analysis of the role of Arp2/3 in mammalian cells, where treatment with CK-666 impaired both mobility and end-resection in human cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDetection of a DSB triggers cell cycle arrest through the DNA damage checkpoint (DDC) for 12\u0026ndash;15 h [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. We found that when Apr2/3 was blocked, cells experienced a 4 h checkpoint which was dependent on the Tel1-Mre11 (TM) checkpoint which arrests cells in response to unresected DSB ends [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Disruption of both the TM checkpoint and Arp2/3 activity prevented cells from entering DSB-induced checkpoint arrest as evidenced by the lack of cells accumulating in G\u003csub\u003e2\u003c/sub\u003e/M arrest and the lack of phosphorylation of Rad53. Arp2/3 was also required to maintain checkpoint arrest as cells began to escape arrest, despite continued Rad53 phosphorylation. Impaired Mec1-mediated checkpoint arrest in budding yeast is consistent with the lower ATR signaling reported in WASP-deficient human cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOf the known Arp2/3 actin nucleation promoting factors in budding yeast, we focused on Las17\u003csup\u003eWASP\u003c/sup\u003e and the type-I myosins Myo3 and Myo5. In humans WASP has been shown to bind to RPA and is implicated in loading RPA onto ssDNA in response to a DSB as mutations in WASP were shown to have less RPA-bound DNA when exposed to the topoisomerase I inhibitor camptothecin (CPT), which causes replication fork collapse [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. We show that the highly conserved C-terminal region across WASP orthologs (WH2 and CA domains) of Las17 is required for DSB mobility. Human WASP binds to RPA, through this highly conserved region and mediates RPA binding to ssDNA [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Impaired RPA binding to ssDNA would prevent Ddc2\u003csup\u003eATRIP\u003c/sup\u003e-mediated Mec1 activation of the DDC.\u003c/p\u003e \u003cp\u003eBudding yeast Myo3 and Myo5 are functionally redundant in endocytosis and deletion of either Myo3 or Myo5 did not affect resection, checkpoint arrest, or repair by gene conversion. However, a deletion of either myosin protein lowered DSB mobility and the insertion of a second copy of Myo5 with its endogenous promotor into a \u003cem\u003emyo3\u003c/em\u003e deletion mutant restored DSB mobility to wildtype levels. Through domain deletion mutants of \u003cem\u003eMYO5\u003c/em\u003e, we found that the SH3 domain of Myo5, which mediates interactions with Vrp1 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], is required for wildtype levels of DSB mobility. Since SH3 domains mediate a wide range of protein-protein interactions, the loss of either type-I myosin might result in an imbalance of interactions required for DSB mobility and the deletion of \u003cem\u003eMYO3\u003c/em\u003e and \u003cem\u003eMYO5\u003c/em\u003e might restore that balance. The loss of both type-I myosins after DSB induction brought DSB mobility back to wildtype levels in a Las17-dependent manner. Deletion of \u003cem\u003eVRP1\u003c/em\u003e impaired the initiation of resection. Since Vrp1 brings Myo3 and Myo5 to Arp2/3 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], the deletion of \u003cem\u003eVRP1\u003c/em\u003e would likely be similar to when both Myo3 and Myo5 are degraded.\u003c/p\u003e \u003cp\u003eChanges in DSB mobility did not necessarily affect overall DSB repair efficiency through HR. While the degradation of Las17 negatively affected the repair efficiency of DSBs through GC and SSA, deletion of either \u003cem\u003eMYO3\u003c/em\u003e or \u003cem\u003eMYO5\u003c/em\u003e did not affect the repair efficiency of DSBs, even when the donor location was moved to unfavorable locations. This difference in repair efficiencies among these mobility deficient mutants might be due to the length of G2/M arrest imposed by the DDC. Since \u003cem\u003emyo3Δ\u003c/em\u003e and \u003cem\u003emyo5Δ\u003c/em\u003e mutants did not affect the length of checkpoint arrest, these mutants had the same amount of time to repair the break as the wildtype. The shortened checkpoint arrest when Las17 was degraded might have triggered an early adaptation response.\u003c/p\u003e \u003cp\u003eBy using single particle tracking and MSD analysis of a single DSB in the \u003cem\u003eMAT\u003c/em\u003e locus on chromosome III in budding yeast, we were able to show that Arp2/3 has a conserved role the DNA damage response and plays an important part in the initiation and facilitation of the resection of DSB ends. The lower DSB mobility that we observe when Arp2/3 activity is blocked, either through drug treatment or degradation of Las17\u003csup\u003eWASP\u003c/sup\u003e, is likely linked to a lower rate of resection, as DSBs in other resection mutants, such as a \u003cem\u003eFUN30\u003c/em\u003e deletion or an \u003cem\u003eexo1 dna2\u003c/em\u003e double deletion, are less mobile than their wildtype counterparts. The rate of resection is not the only determinant of DSB mobility as a deletion of either Myo3 or Myo5 is sufficient to lower DSB mobility but did not affect the rate of resection. Preventing resection triggered a shortened checkpoint arrest through the TM checkpoint and stopping continued resection allowed cells to escape checkpoint arrest early.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eKEY RESOURCES TABLE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRefer to excel file for tables\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods Details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain and plasmid construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll strains are derived from JKM179 [15] which is a well characterized strain with the \u003cem\u003eHML\u0026nbsp;\u003c/em\u003eand \u003cem\u003eHMR\u0026nbsp;\u003c/em\u003edonor domains deleted. Ddc2 and Rad51 were GFP-tagged with a 13 amino acid linker GGSGGSRIPGLIN-eGFP as previously described [13]. Ddc2-GGSGGSRIPGLIN-eGFP and Rad51-GGSGGSRIPGLIN-eGFP are referred to as Ddc2-GFP and Rad51-GFP respectively in this study. All AID-tagged mutants strains were derived from a modified version of JKM179 expressing osTIR1 at URA3 created by cutting pNHK53 [23] with \u003cem\u003eStu\u003c/em\u003eI and integrating into the genome. For degron tagged derivatives, PCR products were generated with mixed oligoes with homology to the C-terminal end and plasmid pJH2892 or pJH2899 [22] to create an 9xMyc-AID (AID) inserts with the KAN or NAT marker. Myo5-domain deletion plasmids [25] were digested with BamHI/SacI to excise a linear fragment for genome integration. Deletion of ORFs, \u003cem\u003emyo5\u003c/em\u003e mutants, and AID tags were introduced with the one step PCR homology cassette amplification and the standard yeast transformation method [58]. Las17 domain deletion mutants were created by CRISPR/Cas9 as previously described [59] with material listed in supplemental tables. Transformants were verified by PCR and western blot. Strains are listed in \u003cstrong\u003eTable S3\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrains containing degron fusions and galactose-inducible HO were cultured using standard procedures. \u0026nbsp;Briefly, a single colony grown on a YPD plate was inoculated in 5ml YP-lactate for ~15 hours overnight at 30\u0026deg; C with agitation. \u0026nbsp;The following day, 500-100ml of YP-lactate was inoculated with the starter culture so that the cell density reached OD\u003csub\u003e600\u003c/sub\u003e = 0.5 the following day after overnight growth at 30 \u0026deg; C with agitation. \u0026nbsp; Once cultures reached the appropriate density, HO expression was induced by addition of 20% galactose to 2% vol/vol final concentration. \u0026nbsp;For auxin treatment, indole-3-acetic acid (IAA) was resuspended in ethanol to a working concentration of 500 mM. For AID degradation cultures were split at the indicated timepoints to add auxin, IAA was added to one culture to a final concentration of 1mM either 1 h before or 2 h after adding galactose. \u0026nbsp;The equivalent volume of 100% ethanol was added to the second culture. \u0026nbsp;Cells were harvested at the indicated timepoint by centrifugation at 3000x for 3 minutes and prepared for microscopy, resection assays, and western blot analysis. Cells prepared for microscopy were washed 3 times with a Leu- media.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlating assays\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe efficiency of DSB repair by homologous recombination or single-strand annealing was determined as previously described for YJK17\u0026nbsp;[48]. Briefly, cells were selected from a single colony on YPD plates and grown overnight in 5ml of YEP-lactate. Cells were diluted to OD\u003csub\u003e600\u003c/sub\u003e = 0.2 and allowed to grow until OD\u003csub\u003e600\u003c/sub\u003e = 0.5-1.0. Approximately 100 cells from each culture were then placed on YEP-Gal (2% gal per volume) or YEP-Gal+IAA (1mM) and YPD in triplicate and incubated at 30\u003csup\u003eO\u003c/sup\u003eC until visible colonies formed. Viability was calculated by dividing the number of colonies on YEP-Gal or YEP-Gal+IAA plates by the number of colonies on YPD plates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLive cell microscopy and mean squared displacement analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLive cell microscopy was performed using a spinning disc confocal microscope using Nikon Elements AR software on a Nikon Ni-E upright microscope equipped with a 100\u0026times; (NA, 1.45) oil immersion objectives, a Yokogawa CSU-W1 spinning-disk head, and an Andor iXon 897U EMCCD camera. Fluorophores were excited at 488nm (GFP) and 561nm (mCherry). Time-lapse series were acquired with 15 optical slices of 0.3\u0026micro;m thickness every 30 seconds for 20 minutes. Time-lapse image stacks were analyzed using a custom MATLAB program designed by the Bloom lab as previously described\u0026nbsp;[60]. Coordinates of DSB and the SPB were tracked with Speckle Tracker, a custom MATLAB program\u0026nbsp;[61, 62]. A custom PERL script was used to convert the pixels to nanometers and subtract the distance of the SPB from the Ddc2-GFP coordinates to eliminate cell and nuclear motion, subtract the mean position of the corrected GFP coordinates, calculate the MSD of each time lapse and export MSD coordinates and radius of confinement (R\u003csub\u003ec\u003c/sub\u003e) to an Excel spreadsheet\u0026nbsp;[60]. In MATLAB spot positions were fitted to [\u0026micro;\u003csub\u003ex\u003c/sub\u003e, \u0026sigma;\u003csub\u003ex\u003c/sub\u003e] = normfit (x \u0026ndash; x\u003csub\u003emean\u003c/sub\u003e) and [\u0026micro;\u003csub\u003ey\u003c/sub\u003e, \u0026sigma;\u003csub\u003ey\u003c/sub\u003e] = normfit (y \u0026ndash; y\u003csub\u003emean\u003c/sub\u003e). The variance of the distribution of spot position was then calculated as \u0026sigma;\u003csup\u003e2\u003c/sup\u003e = mean (\u0026sigma;\u003csub\u003ex\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e, \u0026sigma;\u003csub\u003ey\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e). The average squared deviation from the mean position is (∆r\u003csub\u003e0\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e) = (∆x\u003csub\u003e0\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e) + (∆y\u003csub\u003e0\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e). Using \u0026sigma;\u003csup\u003e2\u003c/sup\u003e and (∆r\u003csub\u003e0\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e), we calculated R\u003csub\u003ec\u003c/sub\u003e as \u003cimg src=\"data:image/png;base64,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\" width=\"147\" height=\"38\"\u003e\u0026nbsp;Due to variations in day-to-day collection, wildtype data was only compared to data collected on the same day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis for Rc comparison was done in PRISM using a t-test or one-way ANOVA analysis. For one-way ANOVA analysis, Dunnett test was used to correct for multiple comparisons. Wildtype comparisons were conducted with data collected from the same day.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResection and cutting assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResection was measured by quantitative PCR (qPCR) analysis using a restriction enzyme digest as previously described\u0026nbsp;[29]. Briefly, cells are grown in YEP-Lac as described above. 50ml of culture was harvested and DNA extracted using a DNA extraction kit (Masterpure Yeast DNA Purification Kit Cat# MPY80200). DNA was diluted to 10ng/\u0026micro;l. Sty1-HF (digest) or an equivalent amount of water (mock) in Cutsmart buffer at 37C\u003csup\u003eO\u003c/sup\u003e for 4 h. qPCR samples were run in triplicate on a Biorad CFX384 Real-Time System C1000 Touch Thermal Cycler qPCR machine using Bio-Rad CFX Maestro 1.1 Version 4.1.2433.1219. \u003cem\u003eADH1\u003c/em\u003e was used as a control gene. See \u003cstrong\u003eTable S4\u003c/strong\u003e for primers. Resection was calculated measuring the fraction of cells that had passed the Sty1 restriction site (RS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" height=\"66\" width=\"257\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ef\u0026nbsp;\u003c/em\u003e is the fraction of cells where HO has been cleaved. \u003cem\u003eE\u003csub\u003eRS\u003c/sub\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eE\u003csub\u003eADH1\u0026nbsp;\u003c/sub\u003e\u003c/em\u003eare the primer efficiencies for the primer pairs 0.7kb, 5kb, 10kb away from the HO-cut site and the \u003cem\u003eADH1\u003c/em\u003e primers. ∆C\u003csub\u003eq\u003c/sub\u003e (digest\u0026ndash;mock) is the difference between quantification cycles between the mock and digested samples.\u003c/p\u003e\n\u003cp\u003eFor cutting assays, DNA was collected as described above. DNA was diluted to 10ng/\u0026micro;l and run using primers flanking the HO-cut site in the \u003cem\u003eMAT\u0026nbsp;\u003c/em\u003elocus on chromosome III. \u003cem\u003eADH1\u003c/em\u003e was used as a control. Gal-HO cutting was measured by the fold increase with the following equation using the 0 h timepoint as a control.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" height=\"22\" width=\"284\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTCA protein extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein extracts were prepared for western blot analysis by the standard TCA protocol described in\u0026nbsp;[63]. Briefly, 15-10ml of harvested cells were incubated on ice in 1.5ml microcentrifuge tubes with 20% TCA for 20 minutes. Cells were washed with acetone and the pellet was air dried. 200ul of MURBs buffer (50mM sodium phosphate, 25mM MES, 3M urea, 0.5% 2-mercaptoethanol, 1 mM sodium azide, and 1% SDS) was added to each sample allow with acid washed glass beads. Cells were lysed by mechanical shearing with glass beads for 2 minutes. Supernatant was collected by poking a hole in the bottom of the 1.5ml microcentrifuge tube and spun in a 15ml conical tube. Samples were boiled at 95C for 10 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDenatured protein samples prepared by TCA extraction were centrifuged at max speed for 1 minute and 8 \u0026ndash; 20 \u0026micro;l of samples were loaded into a 10% or 8% SDS page gel. \u0026nbsp; Proteins were separated by applying 90 V constant until the 37 kDa marker reached the bottom of the gel. \u0026nbsp; Gels were then transferred to an Immun-Blot PVDF using a wet transfer apparatus set to 100 V constant voltage for 1 h. The resulting membranes were blocked in 5% nonfat dry milk or OneBlock buffer (Genesee Scientific, 20-313) for 1 h at room temperature or overnight at 4 \u0026deg;C with gentle agitation. \u0026nbsp;After washing 3 times with 1x TBS-T, blots were incubated with either mouse anti-Myc [9E11] (Abcam, ab56) to detect Tir1 and AID fusions, rabbit anti-Rad53 (Abcam, ab104232), or mouse anti-Pgk1 antibody (Abcam Cat# ab113687, RRID:AB_10861977) for 1 h at room temperature. \u0026nbsp;Blots were then washed 3 times with 1x TBS-T and incubated with anti-mouse or anti-rabbit HRP secondary antibody for 1 h at room temperature. \u0026nbsp; After washing 3 times with 1x TBS-T, ECL Prime was added to fully coat the blots and left to incubate for 5 min at room temperature with gentle agitation. \u0026nbsp;Blots were imaged using a BioRad ChemiDoc XR+ imager and prepared for publication using Image Lab software (BioRad) and Adobe Photoshop CC 2017.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Kerry Bloom for training in MSD measurements, Bruce Goode for donation of CK-666 and expertise, Helle Ulrich for donations of the AID and \u003cem\u003eTIR1\u003c/em\u003e plasmids, David Drubin for donations of the Myo5 plasmids and Susan Gasser for donation of strains. Research was supported by NIH grant R35 GM127029. F.Y.Z, M.A., N.A., K.C., and K.B.F. were supported by NIH Genetics Training Grant\u0026nbsp;TM32GM007122.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization and initial data collection was conducted by Felix Y. Zhou and James E. Haber. Data collection, imaging, primer design, data analysis, and strain making was conducted by Felix Y. Zhou, Marissa Ashton, Yiyang Jiang, Neha Arora, Kevin Clark, and Kate B. Fitzpatrick. The manuscript was authored and edited by Felix Y. Zhou and James E. Haber.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLEAD CONTACT AND MATERIALS AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for reagents may be directed to, and will be fulfilled by the corresponding author, Dr. James E. Haber ([email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHaber JE (2016) A Life Investigating Pathways That Repair Broken Chromosomes. Annu Rev Genet 50:1\u0026ndash;28\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDion V et al (2012) Increased mobility of double-strand breaks requires Mec1, Rad9 and the homologous recombination machinery. Nat Cell Biol 14(5):502\u0026ndash;509\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMine-Hattab J, Rothstein R (2012) Increased chromosome mobility facilitates homology search during recombination. Nat Cell Biol 14(5):510\u0026ndash;517\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeeber A, Hauer MH, Gasser SM (2018) Chromosome Dynamics in Response to DNA Damage. Annu Rev Genet 52:295\u0026ndash;319\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaber JE (2018) DNA Repair: The Search for Homology. BioEssays 40(5):e1700229\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaridi CP et al (2018) Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature 559(7712):54\u0026ndash;60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchrank BR et al (2018) Nuclear ARP2/3 drives DNA break clustering for homology-directed repair. Nature 559(7712):61\u0026ndash;66\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoode BL, Eskin JA, Wendland B (2015) Actin and endocytosis in budding yeast. Genetics 199(2):315\u0026ndash;358\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSymons M et al (1996) Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization. Cell 84(5):723\u0026ndash;734\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor MD et al (2010) Nuclear role of WASp in the pathogenesis of dysregulated TH1 immunity in human Wiskott-Aldrich syndrome. Sci Transl Med 2(37):37ra44\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan SS et al (2022) WASp modulates RPA function on single-stranded DNA in response to replication stress and DNA damage. Nat Commun 13(1):3743\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalletta BJ, Chuang DY, Cooper JA (2008) Distinct roles for Arp2/3 regulators in actin assembly and endocytosis. PLoS Biol 6(1):e1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaterman DP et al (2019) Live cell monitoring of double strand breaks in S. cerevisiae. PLoS Genet 15(3):e1008001\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Dudko OK (2016) First-Passage Processes in the Genome. Annu Rev Biophys 45:117\u0026ndash;134\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SE et al (1998) Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94(3):399\u0026ndash;409\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee CS et al (2014) Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break. Nat Struct Mol Biol 21(1):103\u0026ndash;109\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi K et al (2020) Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break. Proc Natl Acad Sci U S A 117(35):21354\u0026ndash;21363\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoore JK, Haber JE (1996) Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol 16(5):2164\u0026ndash;2173\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIdrissi FZ et al (2012) Ultrastructural dynamics of proteins involved in endocytic budding. Proc Natl Acad Sci U S A 109(39):E2587\u0026ndash;E2594\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheblal A et al (2020) DNA Damage-Induced Nucleosome Depletion Enhances Homology Search Independently of Local Break Movement. Mol Cell 80(2):311\u0026ndash;326e4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobertson AS et al (2009) The WASP homologue Las17 activates the novel actin-regulatory activity of Ysc84 to promote endocytosis in yeast. Mol Biol Cell 20(6):1618\u0026ndash;1628\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorawska M, Ulrich HD (2013) An expanded tool kit for the auxin-inducible degron system in budding yeast. Yeast 30(9):341\u0026ndash;351\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishimura K et al (2009) An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6(12):917\u0026ndash;922\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinter D, Lechler T, Li R (1999) Activation of the yeast Arp2/3 complex by Bee1p, a WASP-family protein. Curr Biol 9(9):501\u0026ndash;504\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewellyn EB et al (2015) An Engineered Minimal WASP-Myosin Fusion Protein Reveals Essential Functions for Endocytosis. Dev Cell 35(3):281\u0026ndash;294\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEvangelista M et al (2000) A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex. J Cell Biol 148(2):353\u0026ndash;362\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeli MI, Riezman H (1996) Role of type I myosins in receptor-mediated endocytosis in yeast. Science 272(5261):533\u0026ndash;535\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoodson HV et al (1996) Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton. J Cell Biol 133(6):1277\u0026ndash;1291\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGnugge R, Oh J, Symington LS (2018) Processing of DNA Double-Strand Breaks in Yeast. Methods Enzymol 600:1\u0026ndash;24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZierhut C, Diffley JF (2008) Break dosage, cell cycle stage and DNA replication influence DNA double strand break response. EMBO J 27(13):1875\u0026ndash;1885\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen H, Lisby M, Symington LS (2013) RPA coordinates DNA end resection and prevents formation of DNA hairpins. Mol Cell 50(4):589\u0026ndash;600\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCostelloe T et al (2012) The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection. Nature 489(7417):581\u0026ndash;584\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEapen VV et al (2012) The Saccharomyces cerevisiae chromatin remodeler Fun30 regulates DNA end resection and checkpoint deactivation. Mol Cell Biol 32(22):4727\u0026ndash;4740\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Z et al (2008) Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134(6):981\u0026ndash;994\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMimitou EP, Symington LS (2008) Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455(7214):770\u0026ndash;774\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLydeard JR et al (2010) Sgs1 and exo1 redundantly inhibit break-induced replication and de novo telomere addition at broken chromosome ends. PLoS Genet 6(5):e1000973\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroelly FJ et al (2023) Targeting DNA damage response pathways in cancer. Nat Rev Cancer 23(2):78\u0026ndash;94\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaterman DP, Haber JE, Smolka MB (2020) Checkpoint Responses to DNA Double-Strand Breaks. Annu Rev Biochem 89:103\u0026ndash;133\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMantiero D et al (2007) Dual role for Saccharomyces cerevisiae Tel1 in the checkpoint response to double-strand breaks. EMBO Rep 8(4):380\u0026ndash;387\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePellicioli A et al (2001) Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest. Mol Cell 7(2):293\u0026ndash;300\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLimbo O et al (2011) Mre11 nuclease activity and Ctp1 regulate Chk1 activation by Rad3ATR and Tel1ATM checkpoint kinases at double-strand breaks. Mol Cell Biol 31(3):573\u0026ndash;583\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez Y et al (1999) Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science 286(5442):1166\u0026ndash;1171\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDubrana K et al (2007) The processing of double-strand breaks and binding of single-strand-binding proteins RPA and Rad51 modulate the formation of ATR-kinase foci in yeast. J Cell Sci 120(Pt 23):4209\u0026ndash;4220\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou L, Elledge SJ (2003) Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300(5625):1542\u0026ndash;1548\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLonghese MP et al (1998) DNA damage checkpoint in budding yeast. EMBO J 17(19):5525\u0026ndash;5528\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDotiwala F et al (2010) Mad2 prolongs DNA damage checkpoint arrest caused by a double-strand break via a centromere-dependent mechanism. Curr Biol 20(4):328\u0026ndash;332\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUsui T, Ogawa H, Petrini JH (2001) \u003cem\u003eA DNA damage response pathway controlled by Tel1 and the Mre11 complex.\u003c/em\u003e Mol Cell, 7(6): pp. 1255-66\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JA, Haber JE (2009) Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete. Proc Natl Acad Sci U S A 106(4):1151\u0026ndash;1156\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee CS et al (2016) Chromosome position determines the success of double-strand break repair. Proc Natl Acad Sci U S A 113(2):E146\u0026ndash;E154\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaze MB et al (2002) Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase. Mol Cell 10(2):373\u0026ndash;385\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGautreau AM et al (2022) Nucleation, stabilization, and disassembly of branched actin networks. Trends Cell Biol 32(5):421\u0026ndash;432\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClerici M et al (2005) The Saccharomyces cerevisiae Sae2 protein promotes resection and bridging of double strand break ends. J Biol Chem 280(46):38631\u0026ndash;38638\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClerici M et al (2008) The Yku70-Yku80 complex contributes to regulate double-strand break processing and checkpoint activation during the cell cycle. EMBO Rep 9(8):810\u0026ndash;818\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShroff R et al (2004) Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr Biol 14(19):1703\u0026ndash;1711\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUsui T, Petrini JH (2007) The Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 directly influence the DNA damage-dependent functions of Rad53. Proc Natl Acad Sci U S A 104(8):2797\u0026ndash;2802\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson BL et al (1998) The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization. J Cell Biol 141(6):1357\u0026ndash;1370\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Martin AC, Drubin DG (2006) Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity. Dev Cell 11(1):33\u0026ndash;46\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWach A et al (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10(13):1793\u0026ndash;1808\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGallagher DN et al (2020) A Rad51-independent pathway promotes single-strand template repair in gene editing. PLoS Genet 16(10):e1008689\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawrimore J et al (2017) Microtubule dynamics drive enhanced chromatin motion and mobilize telomeres in response to DNA damage. Mol Biol Cell 28(12):1701\u0026ndash;1711\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan X et al (2012) The coupling between sister kinetochore directional instability and oscillations in centromere stretch in metaphase PtK1 cells. Mol Biol Cell 23(6):1035\u0026ndash;1046\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan X et al (2009) Protein architecture of the human kinetochore microtubule attachment site. Cell 137(4):672\u0026ndash;684\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller-Fleming L et al (2014) Detection of Saccharomyces cerevisiae Atg13 by western blot. Autophagy 10(3):514\u0026ndash;517\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"double-strand break (DSB) mobility, DSB repair, Arp2/3, actin, myosin, Las17WASP, homology search, DSB end resection","lastPublishedDoi":"10.21203/rs.3.rs-5125308/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5125308/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUsing budding yeast, \u003cem\u003eS cerevisiae\u003c/em\u003e, we show that Arp2/3 actin branching complex has an evolutionarily conserved role in promoting chromosome mobility of double-strand breaks (DSBs). Disrupting Arp2/3 activity after DSB induction, by drug treatment with CK-666 or auxin-induced degron depletion of nucleation promoting factors Las17\u003csup\u003eWASP\u003c/sup\u003e or the type-1 myosins (Myo3 and Myo5), markedly reduced the radius of confinement of DSBs. Arp2/3 was found to be required to initiate and maintain 5\u0026rsquo;to 3\u0026rsquo; resection of DSB ends. Conversely, inhibiting Exo1 and Dna2- dependent long-range resection or deleting \u003cem\u003eFUN30\u003c/em\u003e impaired the mobility of the DSB, while overexpression of Exo1 suppressed resection inhibition by CK-666. Inactivation of Arp2/3 before DSB induction triggered a shortened checkpoint arrest through the Tel1\u003csup\u003eATM\u003c/sup\u003e/Mre11 (TM) checkpoint, while inactivation of Arp2/3 after DSB induction caused cells to escape arrest prematurely. Shortened checkpoint arrest correlated with a reduction in repair by interchromosomal homologous recombination. These results suggest that Arp2/3 regulation plays an unanticipated role in the regulation of processing of DSB ends that is correlated with an increase in DSB mobility and DSB repair.\u003c/p\u003e","manuscriptTitle":"Arp2/3 and Type-I myosins control chromosome mobility and end-resection at double-strand breaks in S. cerevisiae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-07 10:16:18","doi":"10.21203/rs.3.rs-5125308/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e9a8a26a-d10b-4884-9f2a-24648f76b6ae","owner":[],"postedDate":"October 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38518381,"name":"Biological sciences/Molecular biology/DNA damage and repair/Double-strand DNA breaks"},{"id":38518382,"name":"Biological sciences/Molecular biology/DNA damage and repair/Homologous recombination"},{"id":38518383,"name":"Biological sciences/Molecular biology/DNA damage and repair/DNA damage response"}],"tags":[],"updatedAt":"2025-08-06T07:09:56+00:00","versionOfRecord":{"articleIdentity":"rs-5125308","link":"https://doi.org/10.1038/s41467-025-62377-7","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-08-05 04:00:00","publishedOnDateReadable":"August 5th, 2025"},"versionCreatedAt":"2024-10-07 10:16:18","video":"","vorDoi":"10.1038/s41467-025-62377-7","vorDoiUrl":"https://doi.org/10.1038/s41467-025-62377-7","workflowStages":[]},"version":"v1","identity":"rs-5125308","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5125308","identity":"rs-5125308","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}