Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis

Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei, but not that of a meiosis-specific Dmc1. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and/or multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphases I and II, suggesting the absence of DNA damage-induced cell cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase I.


Introduction 1
In D-loop, DNA synthesis occurs from 3'-end of invading strand as a 1 primer. When the synthesized DNA strand is ejected from the D-loop (Allers and 2 Lichten 2001;Hunter and Kleckner 2001), the ejected synthesized ssDNA is 3 able to anneal with the complementary ssDNA in the other end of the DSB. 4 Annealing induces the second DNA synthesis to complete the recombination by 5 producing non-crossovers. This pathway is called synthesis-dependent strand 6 annealing (SDSA) (Allers and Lichten 2001). On the other hand, when the newly 7 synthesized DNA is stably bound to the D-loop, ongoing DNA synthesis can 8 extend a D-loop with a large displaced ssDNA, which is able to anneal with 9 ssDNA on the opposite DSB ends. Additional processing of the intermediates 10 leads to the formation of double-Holliday junction (dHJ) (Schwacha and Kleckner 11 1994). dHJs are specifically resolved into crossovers. Importantly, meiotic 12 recombination is tightly coupled with chromosome morphogenesis such as the 13 formation of the synaptonemal complex (SC), a meiosis-specific zipper-like 14 chromosome structure, which juxtaposes homologous chromosomes in near 15

vicinity (Cahoon and Hawley 2016). 16
Srs2 is a 3'-to-5' SF1 helicase related to bacterial UvrD helicase (Rong 17 et al. 1991). Srs2 protein has some distinct functional domains: 3'-5' DNA 18 helicase domain, Rad51-interaction domain, and also  PCNA-binding domains in the C-terminus (Marini and Krejci 2010). Genetic 20 analyses showed positive and negative roles of Srs2 in the recombination 21 (Marini and Krejci 2010). Biochemical studies have demonstrated that purified 22 Srs2 protein can dislodge Rad51 filament on ssDNAs and dramatically inhibits 23 Rad51-joint molecules via direct interaction with Rad51 in vitro (Krejci et al. 24 2003;Veaute et al. 2003). This biochemical activity of Srs2 supports the idea of 25 Srs2 function as an anti-recombinase. The Rad51-dismantling activity of Srs2 is 26 confirmed by in vivo analysis (Sasanuma et al. 2013a). 27 Deletion of SRS2 gene shows different kinds of genetic interaction with 28 mutants deficient in DNA transaction. The srs2Δ is synthetic lethal with a 29 mutation of the SGS1, encoding a RecQ-type DNA helicase. By forming a 30 complex with Top3 and Rmi1, Sgs1 is known to dissolve the dHJ structure into 31 noncrossovers (Cejka et al. 2010;Wu and Hickson 2003). Moreover, the the role of Srs2 in a late stage of the recombination such as the post-invasion 1 step in the recombination. This lethality is thought to be caused by a fatal defect 2 in the resolution of toxic intermediates in the recombination process. This is 3 supported by the fact that the deletion of RAD51 can suppress the lethality of 4 srs2Δ sgs1Δ and srs2Δ rad54Δ mutants (Gangloff et al. 2000;Schild 1995). 5 During mitosis, crossovers should be suppressed when DNA damage is 6 spontaneously introduced, because the crossover between homologous 7 chromosomes and sister chromatids results in the loss of heterozygosity. In 8 contrast, as described above, meiotic recombination must give rise to at least 9 one essential crossover per chromosome, which is fostered by a group of 10 proteins called ZMM (Zip-, Msh-, Mer) (Shinohara et al. 2008). Previous genetic 11 studies showed a role of Srs2 in meiosis (Palladino and Klein 1992;Sasanuma 12 et al. 2013b). However, the molecular defects associated with srs2 deletion in 13 meiosis have not been described in detail. Therefore, it remains elusive how 14 Srs2 regulates meiotic recombination. 15 In this study, we analyzed the role of Srs2 helicase in meiotic 16 recombination, particularly looking at dynamics of its interacting partner, Rad51. 17 We found that, in the absence of Srs2, abnormal DNA damage associated with 18 Rad51 aggregation accumulates during late prophase-I, after the completion of 19 meiotic recombination. The formation of this DNA damage in the srs2 requires 20 meiotic DSB formation, but is independent of chromosome segregation. We also 21 detected thin line-staining of Rad51 connecting between two adjacent Rad51 22 foci in early prophase in the absence of Srs2, which is rarely seen in the wild 23 type. We propose that Srs2 protects chromosomes in late meiotic prophase-I 24 from accumulation of abnormal DNA damage by properly coupling the 25 completion of meiotic recombination with chromosome morphogenesis. 26

Immuno-staining 24
Chromosome spreads were prepared using the Lipsol method as described 25 previously (Shinohara et al. 2000;Shinohara et al. 2003). Immnostaining was 26 conducted as described (Shinohara et al. 2000). Stained samples were 27 observed using an epi-fluorescence microscope (BX51; Olympus, Japan) with a 28 CHEF DR-III apparatus (BioRad) using the field 6V/cm at a 120°angle. As reported previously (Palladino and Klein 1992;Sasanuma et al. 2013a), the 4 srs2 deletion mutant exhibits reduced spore viability of 36.8%, indicating a 5 critical role of this helicase for meiosis (Fig. S1A). This marked reduction of the 6 spore viability is somehow unexpected given a negative role of this helicase in 7 recombination. 8 We also confirmed the kinetics of meiotic progression in srs2Δ strains 9 by DAPI staining. In the wild-type strain, meiosis I started at 5 h after incubation 10 with sporulation medium (SPM) and was sequentially followed by meiosis II. 11 Finally, ~90% of the wild-type cells completed MII at around 8 h (Fig. 1A). In the 12 srs2Δ mutant, the appearance of cells undergoing MI was delayed by ~2 h and 13 ~75% of cells finished MII at 14 h (Fig. 1A). A similar delay was observed for a 14 srs2 mutant in a different strain background previously (Palladino and Klein 15 1992). This indicates a defect during prophase-I in the srs2 mutant. In srs2 cells 16 after sporulation; e.g. 12 h, we often detected fragmented DAPI bodies in a 17 cell/spore (Fig. 1B), indicating a defect in chromosome segregation during the 18 mutant meiosis. 19 20 The srs2Δ mutant showed a defect in meiotic DSB repair 21 We analyzed meiotic recombination defects the srs2 deletion mutant in more 22 detail. First, we checked the repair of meiotic DSBs in the mutant by Southern 23 blotting. DSB formation was monitored at the HIS4::LEU2 locus, an artificial 24 meiotic recombination hotspot in chromosome III (Fig. S1B) (Cao et al. 1990). In 25 wild type, DSB frequencies reached its maximum value at 3 hours of meiosis 26 (~10% of total signals) and then decreased gradually (Fig. S1C, D). The srs2Δ 27 accumulates DSB at higher levels (~20%) with more hyper-resection than wild 28 type and delays the disappearance by ~ 2h. (Fig. S1D), indicating that Srs2 is 29 required for efficient meiotic DSB repair. We also checked the formation of two 30 recombinant species, crossover (CO) and non-crossover (NCO) at the same 31 locus. The srs2Δ reduces both CO and NCO to 52% and 64% of the wild-type 32 levels (at 6 h; Fig. S1C, D), respectively. These show that Srs2 is necessary for efficient formation of meiotic recombinants. This is consistent with previous 1 return-to-growth experiment showing delayed recombinant prototroph formation 2 in the srs2Δ mutant (Palladino and Klein 1992). 3 During meiotic prophase, homologous chromosomes are tightly 4 coupled with the formation of the synaptonemal complex (SC), a zipper-like 5 chromosome structure linking two homologous chromosomes. Zip1 is a 6 component of the central region of SC, which serves as a marker for synapsis 7 (Sym et al. 1993). A defect in meiotic recombination results in defective SC 8 formation. We checked the SC formation in the srs2Δ mutant by 9 immuno-staining analysis of Zip1 on chromosome spreads as well as a 10 meiosis-specific cohesin component, Rec8 (Fig. S1E). We classified three 11 categories according to Zip1 staining; Dotty Zip1 (Class I), partially extended 12 (Class II) and fully-elongated (Class III), which roughly correspond with leptotene, 13 zygotene and pachytene stages, respectively. In wild type, ~66% of nuclei 14 contained full-elongate Zip1 lines at 4 h and Zip1 signal gradually disappeared 15 from chromosomes. In srs2Δ strains, although Zip1 focus-positive nuclei 16 exceeded 80% at 4 h, the proportion of cells with fully-elongated Zip1 was 17 significantly reduced to 13 and 26% at 4 and 5 h, respectively (Fig. S1F). In wild-type cells, dotty signals of both Rad51 and Dmc1 peaked at 4 h of 32 meiosis (Figs. 1C and S2A). The appearance of Rad51 foci in cells lacking Srs2 is slightly delayed, and the disappearance of the foci is delayed relative to 1 wild-type cells (Fig. 1D), consistent with delayed DSB repair in the mutant. 2 Interestingly, after disappearance of Rad51 foci, we observed 3 reappearance of Rad51 staining with a unique structure after 5 h incubation in 4 the srs2Δ mutant (Figs. 1C and S2A). This staining shows clustering of 5 beads-in-line of Rad51 foci, in which 1-5 bright aggregates of Rad51 are 6 connected with each other through thin threads containing Rad51 as well as 7 much simple big aggregation of Rad51 (referred to as Rad51 aggregates) (Fig.  8   1C). The formation of Rad51 aggregates reach a plateau at 6 h, slightly 9 decreases thereafter, but some cells at 10 or 12 h contained Rad51 aggregates 10 In order to know the nature of the late Rad51 foci/aggregates, we also 24 studied the localization of RPA (Rfa2, a middle subunit of RPA) at late prophase 25 I of the srs2Δ mutant. Immuno-staining showed that, in addition to early RPA foci 26 ( Fig. 1E, F), like Rad51-aggregates, aggregate staining of Rfa2 re-appeared at 27 late times of the srs2 meiosis; e.g. 6-10 h (Fig. 1G). Closer examination reveals 28 that RPA also exhibits a long-line like staining (Fig. 1F). The kinetics of Rfa2 29 aggregates in the srs2 mutant is very similar to that of Rad51 (Fig. 1D, G). Some One possibility is that Rad51 aggregates bind to DNA damage in 1 ribosomal DNA (rDNA) region, whose segregation defect is often observed in the 2 recombination defective mutants (Li et al. 2014). We co-stained Rad51 with 3 anti-Nop1, a marker for an rDNA region (Schimmang et al. 1989). As shown in 4 In mitosis, the sgs1 mutation is synthetic lethal with the srs2 mutation, 5 indicating a redundant role of these two helicases (Gangloff et al. 2000). Sgs1 6 helicase, together with Top3 and Rmi1, is known to prevent the formation of the 7 untangled chromosomes. The absence of Sgs1 results in abnormal meiosis 8 divisions due to accumulation of un-resolve recombination products involving Schild 1995). To circumvent this, we used Rad54-anchor away system, which 29 specifically depletes nuclear Rad54 fused with RFB by the addition of the drug 30 rapamycin (Haruki et al. 2008;Subramanian et al. 2016). The srs2 RAD54-RFB 31 cells grow normally in the absence of rapamycin while the srs2 RAD54-RFB 32 cells grow poorly on the plate containing the drug, confirming synthetic lethality of the rad54 and srs2 (Fig. 3A). In order to know the functional relationship 1 between Rad54 and Srs2 during late meiosis, first, we added rapamycin at 4 h to 2 RAD54-RFB and srs2 RAD54-RFB cells and analyzed both spore viability and 3 Rad51 foci. The srs2 RAD54-RFB decreased spore viability to 64% in the 4 absence of the drug. As reported (Shinohara et al. 1997b), RAD54-RFB cells 5 decreased spore viability to 48% in the presence of the drug. Addition of the 6 rapamycin also reduced the spore viability of the srs2 RAD54-RFB to 24%, 7 indicating the additive effect of the srs2 deletion and RAD54 depletion on spore 8 viability (Fig. 3B). RAD54 depletion does not affect delayed MI progression in the 9 srs2 deletion (Fig. 3C). As in wild-type cells, RAD54-RFB cells showed normal 10 assembly and disassembly of Rad51 foci in the absence of the drug (Rapa -; Fig.  11 3D, E). However, we found that, from 5 h, one hour after the addition of the drug 12 (Rapa + ), a new class of Rad51 staining appeared. This class contains 5-10 13 brighter foci of Rad51, called "Rad51 clump", which is distinct from the typical 14 times, the ndt80 mutant showed the reduced number of Rad51 foci compared to 5 early time points (Fig. S3E). However, Rad51 foci seemed to turn over less 6 efficiently in the ndt80 mutant (Fig. S3E, F). Little Rad51 aggregate formation 7 was seen in srs2 ndt80 cells arrested at mid-pachytene both on chromosome 8 spreads and in whole cells (Figs. 4A and S3E). This indicates that the formation 9 of Rad51 aggregates in the srs2 mutant depends on Ndt80, thus after the exit of 10 mid-pachytene stage. 11 When the kinetics of Rad51 aggregate formation in the srs2 mutant was 12 compared to kinetics of meiosis I entry, Rad51 aggregate in the srs2 mutant 13 appear 1 h earlier than the entry into meiosis I (Fig. 1D). To confirm this, we 14 In order to confirm that Rad51-aggregate formation in the srs2 is independent of 30 the onset of anaphase I, we used a meiosis-specific null mutant of the CDC20, 31 which encodes an activator of Anaphase promoting complex/cyclosome 2003), the cdc20-mn shows an arrest at the onset of anaphase I. In the 1 cdc20-mn, Rad51 foci appear and disappear like in wild-type control. As 2 expected from the results with benomyl, the Rad51-aggregate formation occurs 3 after the disappearance of Rad51 foci in the srs2 cdc20-mn double mutant as in 4 the srs2 mutant (Fig. 5D, E). This supports the notion that Rad51-aggregate 5 formation in srs2 mutant is independent of the entry into anaphase-I, thus 6 chromosome segregation. 7 The relationship between the formation of Rad51 aggregates and late 8 meiotic prophase I such as SC disassembly was compared by immuno-staining 9 of Rad51 with Zip1 (Fig. S4A). After the pachytene exit, the central region of SCs 10 is dismantled as seen in the loss of Zip1-line signals from chromosomes (Sym et 11 al. 1993). The srs2 cells containing Rad51 aggregates were almost negative for 12 Zip1 lines (Fig. S4A). 13 We also performed the staining of Red1, which is a component of 14 chromosome axes (Smith and Roeder 1997). Most cells with Rad51 foci at 3-5 h 15 are almost positive for Red1 staining in both cdc20-mn and srs2 cdc20-mn cells 16 (Fig. 5D, E). In contrast, srs2 cdc20-mn cells with Rad51 aggregates were 17 negative for Red1 signal. These indicate that Rad51 aggregate formation in the 18 srs2 occurs after or during disassembly of Red1-axes. This is confirmed in the 19 background of wild type too (Fig. S4B, C). Rec8 line positive spreads contained Rad51 foci (Fig. S4D, E). In srs2 cells with 24 or without cdc20-mn, Rad51 aggregates are predominantly seen in cells with 25 Rec8-dots (Fig. S4D, E). 26

27
The srs2 mutant accumulated bridge staining of Rad51 between two 28 recombination foci during early prophase-I 29 During our staining analysis, we noticed that the srs2 cells show very unique thin 30 line staining of Rad51 during early prophase such as 4 h (Fig. 4B). The thin between two Rad51 foci were observed at ~40% frequency of srs2 spreads at 4 1 h (middle graph of Fig. 4C). A few Rad51 bridges were seen in wild type. We 2 also found the Rad51-bridge staining among more than three Rad51 foci in srs2 3 cells, but not in wild type (right graph of Fig. 4C). Careful examination of Rad51 4 foci in the wild type often detected a Rad51 focus with "single tail (or whisker)" 5 (left graph of Figure 4C). The number of Rad51 tail from a single Rad51 focus is 6 almost one. There is few focus with more than 2 tails. When measured the 7 length of the bridge between two foci, we found both wild-type and the srs2 cells 8 show similar distribution of the lengths (Fig. 4D). These results indicate that Srs2 9 suppresses the formation of Rad51 bridges. Indeed, the srs2 cells increased the 10 frequency of the Rad51 bridges and more connections among more than two 11 foci relative to the wild type (Fig. 4C). 12 We then used super-resolution microscopy to analyze Rad51 localization 13 on meiotic chromosomes at high resolution. A structural illumination microscope 14 (SIM) was used to determine Rad51 localization in wild type and srs2 cells at 4h 15 ( Fig 4E). As shown above, in the srs2 mutant, we detected both Rad51 bridges 16 and tails more than in wild type. The wild type the srs2 mutant shows Rad51 foci 17 with tail/bridge at a frequency of 15.4±4.2% (n=18) and 54.3±8.5% (n=20), 18

respectively. 19
The average length of the bridge is ~0.4 µm (Fig. 4D). If the bridge is 20 postulated to consist of a single Rad51 filament on the ssDNA, which is 21 The srs2Δ mutant shows decreased levels of CO and NCO relative to the 14 wild-type, indicating a positive role of Srs2 in meiotic recombination 15 (pro-recombination role). This weak defect in the recombination is consistent 16 with delayed DSB repair (delayed disassembly of Rad51 foci) as well as 17 defective SC formation in the mutant. Our studies also showed that the srs2 18 mutant is partially defective in a step after the DSB processing. However, this 19 "weak" defect in the recombination cannot explain reduced spore viability of the 20 mutant, since the mutants with 50% reduction of CO show high spore viability; 21 e.g. spo11, xrs2, msh4/5 hypomorphic mutants (Martini et al. 2006;Nishant et al. 22 2010;Shima et al. 2005). Consistent with low spore viability of the mutant, we 23 and others detected abnormal chromosome segregation in srs2 meiosis, 24 suggesting the presence of DNA abnormality in the mutant. 25 In this study, we described "unusual" DNA damage formed in the 26 absence of Srs2 helicase during meiosis. This damage is marked with the 27 association of the recombination protein, Rad51, with a large quantity, which we 28 refer to as "Rad51 aggregate". The Rad51 aggregate is not a protein 29 aggregate since it contains another recombination protein, Rad52, as well as The formation of Rad51 aggregates in srs2Δ mutant requires Spo11 2 catalytic activity, thus DSB formation. On the other hand, kinetic analysis 3 revealed that Rad51 aggregates in srs2Δ mutant appear in late prophase-I after 4 the disappearance of Spo11-dependent Rad51 foci associated with meiotic 5 recombination. Rad51 aggregates appear just after the disappearance of 6 "normal" Rad51 foci. This suggests that the formation of Rad51 aggregates 7 occur after the completion of DSB repair such as Rad51-mediated strand 8 invasion. Consistent with this, the ndt80 mutation, which induces an arrest at 9 mid-pachytene stage, blocks the aggregate formation in the srs2Δ mutant. The One possibility to explain Rad51 aggregate formation in the srs2 mutant 24 is that, after the exit of Ndt80-execution point, there might be unrepaired DSBs, 25 which could be repaired by Rad51-dependent pathway (but not Dmc1-pathway) 26 during late prophase-I. The srs2 mutant might be specifically defective in this 27 DSB repair after the pachytene exit. In this pathway, Srs2 may be essential for 28 Rad51 removal, which may lead to the accumulation of unrepaired ssDNAs. 29 However, this is unlikely since even DSB ends formed during pachytene are 30 bound by Dmc1 as well as Rad51. However, the Rad51 aggregates in the srs2 31 mutant do not contain Dmc1 even when Dmc1 protein is present in a cell. 32 Alternatively, Rad51 aggregates and/or its associated DNA damage are formed in two-step process. Frist, DSB repair in the absence of Srs2 may 1 result in the formation of aberrant recombination products/intermediates such as 2 entangled duplexes DNAs (see Fig. 6B). Second, this aberrant 3 product/intermediate might be converted into DNA damage with Rad51 4 aggregates in late prophase-I. Consistent with this two-step model, we found a 5 novel structure called Rad51-bridge (or whisker), thin lines of Rad51 which 6 connect Rad51 foci. This bridge is seen at early prophase I of the srs2 mutant 7 more frequently than in wild type. 8 The presence of Rad51-bridge and -whisker from Rad51 focus 9 suggests that Rad51 focus is not a simple Rad51 filament, rather may contain a 10 three-dimensional configuration of Rad51 filament (Fig. 6A). Rad51-bridge line 11 staining is reminiscent of anaphase bridge or ultra-fine bridge of chromosomes Given that Rad51-bridge in the srs2 mutant is formed between Rad51 30 foci, Srs2 might play a role of this kind of Rad51-associated DNA entanglement 31 between the two DSB sites (Fig. 6B). One likely intermediate is multiple invasion multiple loci. Therefore, Srs2 might play a role in resolution of multiple-invasion 1 by controlling Rad51 filament dynamics using its Rad51-dismantling activity. 2 Bishop and his colleagues show a pair of Rad51 foci during early 3 meiotic pro-phase I are formed in the two ends of a single DSB site (Brown et al. 4 2015). Thus, it is likely that the Rad51 bridge we observed is formed between a 5 pair of Rad51 foci on the two DSB ends. If so, one likely possibility is that the 6 bridge is a ssDNA between two DSB ends. One way to connect the two DSB 7 ends is bridged by the annealing of ejected ssDNA from the D-loop after the 8 DNA synthesis (Fig. 6B). Since the bridge is mainly seen in the absence of Srs2, 9 we propose that Rad51 dismantling activity of Srs2 promotes the removal of 10 Rad51 from the rejected ssDNA. Moreover, it is likely that Srs2 also remove 11 Rad51 in the other end of the DSB during the second end-capture. This idea 12 could explain the formation of the bridge between two Rad51 foci in the srs2, but 13 not in wild type. In wild-type cells, Srs2 seems to remove Rad51 assembly from 14 the intermediates for the second end capture. Importantly, genetic analysis of 15 mitotic recombination in the srs2 mutant suggest the role of Srs2 to facilitate the 16 annealing of the newly synthesized strand to second resected ends by removing 17 Rad51 from the second end (Elango et al. 2017;Ira et al. 2003;Liu et al. 2017;18 Mitchel et al. 2013). 19 We still cannot figure out recombination products formed in the 20 absence of Srs2, which trigger the formation of the Rad51 aggregate. 2D gel 21 analysis has shown that there is few accumulation of abnormal recombination 22 intermediate such as multiple dHJs (Lichten/Goldman, accompanying paper). 23 Thus, multiple dHJs is unlikely. Rather, there might be an entanglement of DNA 24 strands after the completion of the meiotic recombination (Fig. 6B). This 25 intermediate seems to be related to a lethal recombination intermediate formed 26 in the srs2 mutant during mitosis. 27 In either scenario, our analysis reveals a novel pathway to protect 28 meiotic cells in late prophase-I from the formation of aberrant DNA damage 29 induced by Spo11. This repair pathway heavily depends on Srs2 function. For 30 this function, Srs2 is almost essential for meiosis. We would like to point out that 31 Rad51 aggregates in the srs2 mutant is related to lethal recombination 32 intermediates in mitotic cells with SRS2 deletion, which is postulated to form through two-step model. 1 Rad51 aggregate-associated DNA damage seems unrepaired during 2 meiosis. During late prophase-I, there should be sister chromatid or other 3 recombination partners to repair the damage, this might be due to the presence 4 of Rad51-inhibitor Hed1, which clearly suppresses Rad51-mediated DNA repair. 5 The result that CHEF-Southern for chromosome III did not detect any 6 DNA fragmentation at times of Rad51-aggregates in the srs2Δ mutant; e.g. 6 7 and 8 h, implies Rad51 aggregate-associated DNA damage does not contain 8 DSBs. One simple interpretation is that Rad51 aggregates are on either the 9 ssDNA gaps or unwound duplex DNAs. suggests that DNA damage with Rad51 aggregates in the srs2 is masked by the 31 checkpoint activation or there is no such mechanism in late G2 phase of meiotic the activation of the checkpoint during this phase. 1 2

Role of Rad54 in late recombination 3
Upon Rad54 depletion after the assembly of Rad51 on the ssDNA during 4 meiosis, we found a novel staining of Rad51 called Rad51 clump, which is 5 different from typical Rad51 foci in wild-type and aggregates in the srs2 cells.