Mechanisms restraining break‐induced replication at two‐ended DNA double‐strand breaks
2021; Springer Nature; Volume: 40; Issue: 10 Linguagem: Inglês
10.15252/embj.2020104847
ISSN1460-2075
AutoresNhung Pham, Zhenxin Yan, Yang Yu, Mosammat Faria Afreen, Anna Malkova, James E. Haber, Grzegorz Ira,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoArticle12 April 2021free access Source DataTransparent process Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks Nhung Pham Nhung Pham orcid.org/0000-0003-4792-3761 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Zhenxin Yan Zhenxin Yan Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Yang Yu Yang Yu Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Mosammat Faria Afreen Mosammat Faria Afreen Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA Search for more papers by this author Anna Malkova Anna Malkova Department of Biology, University of Iowa, Iowa City, IA, USA Search for more papers by this author James E Haber James E Haber Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA Search for more papers by this author Grzegorz Ira Corresponding Author Grzegorz Ira [email protected] Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Nhung Pham Nhung Pham orcid.org/0000-0003-4792-3761 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Zhenxin Yan Zhenxin Yan Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Yang Yu Yang Yu Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Mosammat Faria Afreen Mosammat Faria Afreen Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA Search for more papers by this author Anna Malkova Anna Malkova Department of Biology, University of Iowa, Iowa City, IA, USA Search for more papers by this author James E Haber James E Haber Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA Search for more papers by this author Grzegorz Ira Corresponding Author Grzegorz Ira [email protected] Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Author Information Nhung Pham1, Zhenxin Yan1, Yang Yu1, Mosammat Faria Afreen2, Anna Malkova3, James E Haber2 and Grzegorz Ira *,1 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 2Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Waltham, MA, USA 3Department of Biology, University of Iowa, Iowa City, IA, USA *Corresponding author. Tel: +1 713 798 1017; E-mail: [email protected] The EMBO Journal (2021)40:e104847https://doi.org/10.15252/embj.2020104847 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two-ended DNA double-strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single-ended DSBs are repaired by break-induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two-ended DSBs. Here, we demonstrate that BIR is suppressed at two-ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D-loop unwinding helicase Mph1, and (iii) Mre11-Rad50-Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans. Synopsis While mutagenic break-induced replication (BIR) repairs single-ended DNA double-strand breaks (DSBs), it needs to be suppressed at two-ended breaks to ensure their faithful repair via gene conversion. Here, the coordinated usage of both DSB ends by ssDNA annealing and resection pathways is found to restrain unwanted BIR in budding yeast. Rad52- and Rad59-mediated ssDNA annealing suppresses BIR by promoting second-end capture. The Mre11-Rad50-Xrs2 complex suppresses BIR by promoting the synchronous formation of ssDNA at both ends of a break. ssDNA annealing is less important for DSB repair and preventing BIR when homology is long. Repair by BIR is not equally initiated at different genomic loci. Introduction Two-ended double-strand breaks (DSBs) are repaired by either non-homologous end-joining (NHEJ) or homologous recombination (HR). In the most basic HR pathway by gene conversion, one of the DSB ends invades the homologous template, forming a displacement loop (D-loop) that primes short-patch new DNA synthesis. Repair is completed when the newly synthesized strand is unwound and anneals to the second end of the DSB leading to conservative inheritance of newly synthesized strands (Ira et al, 2006) (Fig 1A). This mechanism, called synthesis-dependent strand annealing (SDSA), is used in mitotically growing cells. Alternatively, the second end of a DSB anneals to the extended D-loop leading to the formation of a double Holliday junction (dHJ), an intermediate common in meiotic recombination and essential for crossover outcomes (Symington et al, 2014). In repair of one-ended DSBs or when homology within the genome is present only for one end of the DSB, the single end invades the template and initiates extensive repair-specific DNA synthesis that can reach even the end of the chromosome. This pathway, called break-induced replication (BIR), proceeds via D-loop migration and leads to conservative inheritance of the newly synthesized strands (Fig 1A) (Donnianni & Symington, 2013; Saini et al, 2013). Both SDSA and BIR lead to a significant increase of point mutations along the entire length of new DNA synthesis (McGill et al, 1989; Ponder et al, 2005; Hicks et al, 2010; Deem et al, 2011; Shee et al, 2012; Saini et al, 2013; Sakofsky et al, 2014); however, the mutation load is far greater in BIR because of the increased length of repair-specific and low-fidelity DNA synthesis. In addition, frequent template switches are common in BIR within the first 10 kb of the strand invasion site (Smith et al, 2007; Anand et al, 2014; Stafa et al, 2014). The increase of point mutations during DSB repair is attributed to the exposure of long single-strand DNA (ssDNA). Exposed ssDNA is prone to mutagenesis, and unwinding of the newly synthesized strand from its template decreases mismatch repair efficiency (Saini et al, 2013; Sakofsky et al, 2014). The frequent template switches in BIR are likely due to the intrinsic instability of the D-loop intermediate (Smith et al, 2007; Piazza et al, 2019). In diploids, the extensive use of BIR poses a threat to genome integrity by creating new mutations, template switches, and the loss of heterozygosity. In both diploids and haploids, BIR also generates nonreciprocal translocations by template switching between dispersed homologous repeats (Anand et al, 2014). In humans, BIR is less characterized; but similar to yeast, extensive repair-specific DNA synthesis depends on POLD3 (yeast Pol32) and PIF1 (yeast Pif1), and conservative inheritance of newly synthesized DNA was observed during telomere recombination and during mitotic DNA synthesis (MiDAS) that likely proceeded by BIR (Lydeard et al, 2007; Costantino et al, 2013; Wilson et al, 2013; Bhowmick et al, 2016; Roumelioti et al, 2016; Macheret et al, 2020; Li et al, 2021). Moreover, BIR and the related microhomology-mediated BIR (MMBIR) is likely the mechanisms underlying many forms of genome instability in mammalian cells (Lee et al, 2007; Hastings et al, 2009; Carvalho et al, 2013; Sakofsky et al, 2015; Li et al, 2020). At least some of the nonrecurrent structural variations that involve large copy number gains are accompanied by hypermutations, implicating BIR as the mechanism (Beck et al, 2019). Figure 1. Role of Rad59 and DNA binding domain of Rad52 in suppressing BIR at two-ended DSBs Models of DSB repair by SDSA and BIR. Schematic of the H-150 assay. Blue boxes depict homologous sequence on one end of the DSB and green boxes depict homology on the other end. A DSB is induced at modified MATa locus (Chr. III). Strand invasion occurs within the “Z” sequence (blue box) and after copying Yα-inc (dashed line) and X sequence (150 bp), the X sequence is used to capture the second end during SDSA. When copying continues to the end of chromosome via BIR (dashed line), an acentric chromosome forms and an unrepaired chromosome segment remains, leading to cell death. Representative Southern blots showing DSB repair products in WT, rad59Δ, rad52-R70A, and rad59Δ rad52-R70A cells. DNA was digested with Bsp1286I and probed with a MAT-distal sequence (yellow box in panel B). A detailed map of restriction cut sites and expected size for different repair products are illustrated in Appendix Fig S1. Percentage of BIR products among repair outcomes by 6 h are shown. (Mean ± SD; n = 3). Viability of indicated strains is shown (mean ± SD; n = 3). Graph shows repair efficiency among all cells by 6 h (left) and represents the sum of DSB repair by BIR and SDSA compared to the uncut parental band at 0 h (mean ± SD; n = 3). Graph shows repair by BIR and SDSA at 6 h after break induction in mutant cells compared to repair products in WT, which are set to 100% (right). Schematic of H-150 no-gap assay, where 150 bp of Yα homology on the second end of the break is immediately adjacent to the break site. Representative Southern blots showing DSB repair products in indicated mutants in H-150 no-gap assay. Percentage of BIR product among all repair products by 6 h are shown. (Mean ± SD; n = 3). Viability of indicated strains (mean ± SD; n = 3). Graphs showing repair efficiency compared to uncut parental band (left) (mean ± SD; n ≥ 3) or compared to the repair products in WT (right) at 6 h after DSB induction. See Fig 1F legend for details. Data information: Welch’s unpaired t-test was used to determine the P-value in all panels. Source data are available online for this figure. Source Data for Figure 1 [embj2020104847-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint How cells limit the use of BIR in repair of two-ended breaks is not well understood. When homology on one of two ends of a DSB is short (46–150 bp), BIR contributes to or even outcompetes SDSA (Malkova et al, 2005; Mehta et al, 2017). Also, elimination of the D-loop destabilizing enzyme Mph1, (Sun et al, 2008; Prakash et al, 2009; Stafa et al, 2014; Piazza et al, 2019), likely stabilizes D-loops, resulting in increased BIR outcomes, at least when homology is short (Mehta et al, 2017). Consistently, BIR is suppressed when Mph1 is overexpressed (Luke-Glaser & Luke, 2012; Stivison et al, 2020). Here, we hypothesized that enzymes ensuring coordination of the usage of two DSB ends in repair may prevent BIR. Proteins important for annealing, D-loop unwinding, initial resection, and maintaining two DSB ends together were examined in this study. Annealing of ssDNA in yeast is mediated by Rad52 and Rad59 (Mortensen et al, 1996; Sugiyama et al, 1998). Both purified enzymes show annealing activity, but only Rad52 can anneal complementary ssDNA in the presence of RPA (Petukhova et al, 1999; Wu et al, 2006). Rad59 interacts with Rad52 and stimulates the annealing activity of Rad52 in suboptimal conditions, likely by mitigating the negative impact of Rad51 binding to Rad52 on ssDNA annealing (Davis & Symington, 2001; Wu et al, 2008; Gallagher et al, 2020). In cells, Rad52 is required for single-strand annealing, whereas Rad59 seems to be particularly important for annealing of shorter and not completely homologous sequences (Sugawara et al, 2000). Purified yeast or human Rad52 mediates second end capture to an extended joint molecule (McPherson et al, 2004; McIlwraith et al, 2005; Sugiyama et al, 2006; McIlwraith & West, 2008; Nimonkar et al, 2009). Analysis of recombination intermediates in meiosis also supports the later role of Rad52-mediated annealing in both crossover and noncrossover pathways (Lao et al, 2008). Here, we tested whether the elimination of the annealing activity, that is required for the second end capture to form double Holliday junctions or to complete SDSA, can unleash BIR during repair of two-ended breaks. Besides the role of “annealases” in controlling BIR during repair of two-ended DSBs, we also tested the possible function of Mph1 which disrupts the extended D-loop in the SDSA pathway (Sun et al, 2008; Prakash et al, 2009), and the Mre11-Rad50-Xrs2 complex (MRX) responsible for initial end resection (reviewed in (Symington, 2016)) and maintaining two ends of a DSB together (Kaye et al, 2004; Lobachev et al, 2004). Finally, we examined the effect of the chromatin state of the donor template on the competition between BIR and SDSA. Multiple intra- and interchromosomal DNA recombination assays with a broad range of homology sizes (150 bp to over 100 kb) were used to test the function of these enzymes in the competition between SDSA and BIR. Results Rad59- and Rad52-mediated ssDNA annealing suppresses BIR during repair of DSB Initial steps of both SDSA and BIR involve strand invasion of one DSB end that results in formation of a displacement loop (D-loop). Gene conversion via SDSA is completed when the D-loop is extended, and the newly synthesized strand is displaced from its template and anneals to the second DSB end (Fig 1A). In BIR, the D-loop migrates even to the end of the chromosome, creating initially a long ssDNA strand that is then converted into a double-stranded product (Saini et al, 2013). Previous work showed that 46, 50 bp, or even 150 bp homology on the second end is too short for efficient second end capture and for completion of SDSA, resulting in high level of BIR (Deem et al, 2008; Mehta et al, 2017). In these ectopic recombination assays, an additional potential impediment for second end capture was the presence of ~700 bp non-homologous gap between homologous sequences (Mehta et al, 2017). Here, we have used a number of new assays with much longer homology ranging from 150 bp to over 100 kb, and assays with or without the gap to study the role of proteins mediating annealing and other enzymes in the competition between SDSA and BIR for DSB repair. We hypothesized that decreased annealing activity would prevent the completion of gene conversion and allow the D-loop to extend further to finish the repair by BIR. To test the role of ssDNA annealing in the choice of repair pathway between BIR and SDSA at two-ended DSBs, a separation-of-function mutant rad52-R70A was introduced. This mutant is severely defective in DNA binding and ssDNA annealing, but capable of loading Rad51 properly to initiate strand exchange (Shi et al, 2009). Additionally, rad59Δ and rad59Δ rad52-R70A double mutants were constructed. All these mutants were previously shown to reduce the SSA repair pathway which relies entirely on ssDNA annealing (Ivanov et al, 1996; Mortensen et al, 1996; Sugiyama et al, 1998; Petukhova et al, 1999; Davis & Symington, 2001; Wu et al, 2006; Gallagher et al, 2020). We initially used a previously developed assay, a modified intrachromosomal mating-type switching to study competition between SDSA and BIR (Mehta et al, 2017). A DSB is induced by HO endonuclease at the MATa locus and repaired by recombination with an HMLα-inc template, the resulting MATα-inc product is not cleaved by HO endonuclease. The second template for mating-type switching, HMRa, was deleted. The difference between native MAT switching and this recombination assay is that the homology size of the second DSB end is shortened from ~1,400 bp to 150 bp (H-150 assay) (Fig 1B). The successful capture of the short second end leads to gene conversion of MATa to MATα-inc and produces viable colonies. In the event that the second end is not captured, DNA synthesis via migrating D-loop (BIR) continues to the end of the chromosome (12 kb distance), including an apparent crossover: HML/MATα-inc. The BIR product is inviable, as BIR forms an acentric chromosome fragment and leaves unrepaired the chromosome fragment carrying the centromere. SDSA and BIR products can be distinguished by different restriction fragment sizes on a Southern blot (Fig 1C). A detailed map of restriction cut sites and expected sizes for different repair products (BIR, SDSA, and crossovers) are illustrated in Appendix Fig S1. We note that crossovers accompanying gene conversion do not contribute to repair in this assay, because we do not observe the reciprocal crossover product. As previously shown in this H-150 assay in wild-type cells, SDSA dominated with ~82% contribution to DSB repair, with the remaining 18% being BIR products. Eliminating Rad59 reduced the contribution of the SDSA pathway to ~13%, while in rad52-R70A or rad59Δ rad52R70A mutants, SDSA was further reduced to ~1% (Fig 1C and D). We note that while an important role of Rad59 and Rad52R70 in SDSA was shown previously, the contribution of BIR had not been tested (Bai et al, 1999). A switch to BIR is consistent with a marked decrease in the mutants’ viability, as BIR is a lethal event in this assay (Fig 1E). However, not all SDSA events were channeled to BIR in the annealing mutants, as overall product formation (BIR + SDSA) 6 h after break induction was decreased when compared to wild-type cells (Fig 1F). This finding suggests that either BIR is decreased or delayed in annealing mutants, or that short 150 bp homology on the second end interferes with BIR. Both of these possibilities are tested below. Besides the well-established role of Rad52 in loading Rad51 and ssDNA annealing, Rad52 and its DNA-binding domain also negatively regulate extensive resection, particularly in fission yeast (Yan et al, 2019). It is unlikely that the role of Rad52 in resection suppresses BIR, because the rad52-R70A mutant shows only a minor increase in the rate of extensive resection, much less than that observed in the equivalent fission yeast DNA-binding mutant rad52-R45A (Appendix Fig S2A and Yan et al, 2019). Second, the deletion of DOT1 that causes a ~2-fold increase in extensive resection rate (Chen et al, 2012) does not alter SDSA/BIR contribution to DSB repair (Appendix Fig S2B). Finally, we note that in rad52Δ or rad51Δ strains, no repair by BIR or SDSA was observed (Appendix Fig S2C). In the mating-type switching assay and its derivative H-150 (Fig 1B), the Ya sequence is replaced by Yα-inc. Because the Ya sequence is non-homologous with the template, the invading strand must synthesize across about 700-bp Yα-inc gap to reach 150 bp homology with the second end. It is possible that this gap and/or the non-homologous tail on the resected DSB end interferes with engaging the second end in recombination. To eliminate these constraints, we tested recombination between an HMLα-inc and truncated MATα, where only 150 bp of Yα remains to the left of the DSB. In this assay, homology is present immediately at both DSB ends (H-150 no-gap assay, Fig 1G, Appendix Fig S1). In wild-type cells, SDSA dominated with > 95% of total DSB repair, while in annealing-defective rad59Δ and rad52-R70A mutants, SDSA dropped to ~55 and 1%, respectively, while BIR increased (Fig 1H–K). The double mutant rad59Δ rad52-R70A showed a similar phenotype as the rad52-R70A single mutant. Similar to the H-150 assay, the overall repair efficiency decreased in annealing mutants (Fig 1K). Together, these results confirm that annealing activity suppresses BIR at DSBs with short homology, either with or without a non-homologous gap between homologous sequences. By comparing results obtained in H-150 assays with or without the gap, we also conclude that the gap and/or the presence of a non-homologous tail likely interfere with the second end capture in SDSA and therefore increase the usage of BIR, at least when homology on the second end is short (Appendix Fig S3). BIR mildly decreases in annealing-defective mutants To test whether BIR itself is affected by any of the annealing mutations, we constructed a new system, called H-0, where all homology to HMLα-inc on the second end of the break was eliminated (Fig EV1A). As confirmed by Southern blot, BIR is the sole pathway of DSB repair and cells do not survive, as BIR is lethal in this system (Fig EV1B and C). In annealing-defective rad52-R70A mutant strain, we observed a decrease of BIR product formation 6 h after DSB induction to ~60% of the wild-type levels (Fig EV1D). Again, the double mutant rad59Δ rad52-R70A showed a similar phenotype as rad52-R70A single mutant. These results suggest that BIR tested here is mildly deficient in annealing-defective mutants. Click here to expand this figure. Figure EV1. BIR efficiency is mildly decreased in annealing mutants Schematic of the H-0 BIR assay. DSB is induced at a modified MATa locus (Chr. III). Strand invasion occurs within the “Z” sequence (blue box) and DNA synthesis continues to the end of the chromosome. Representative Southern blots showing DSB repair products in WT, rad59Δ, rad52-R70A, and rad59Δ rad52-R70A cells. DNA was digested with Bsp1286I and probed with a MAT-distal sequence (yellow box in panel A). Viability of indicated strains is shown (mean ± SD; n = 3). Repair efficiency compared to parental MATa at time 0 h (left) and repair efficiency of indicated mutants compared to WT by 6 h (right) (mean ± SD; n ≥ 3). Representative Southern blots showing BIR repair product in H-0 system in WT and indicated mutant cells. Repair efficiency compared to parental MATa at time 0 h (left) and repair efficiency of indicated mutants compared to WT by 6 h (right) (mean ± SD; n ≥ 3). Model presenting the possible function of DNA binding/annealing domain of Rad52 in stabilizing the D-loop. Rad52 stabilizes/extends D-loop by three-strand exchange (left) or Rad52 anneals a 3′ invading strand unwound from its template back to disrupted, but still RPA-bound, D-loop (right). Data information: Welch’s unpaired t-test was used to determine the P-value in all panels. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns). Download figure Download PowerPoint In the H-0 assay, while there is no homology on the second end of the break, the homology on the invading end is also limited to ~300 bp. We therefore tested the role of annealing in the BIR assay where the invading end has no homology constraint. We employed an established BIR assay that involves recombination between a truncated and a complete chromosome III, where one DSB end has an extensive homology (~200 kb) with the template but the centromere-distal end shares only 46 bp homology (Fig EV2A) (Malkova et al, 2005). In this disomic system, there was no loss of viability after HO induction in the mutant strains. Approximately 80% of the wild-type cells use BIR for repair, during which the break end with extensive homology invades into the homologous chromosome and copies over 100 kb to the end of the chromosome. The remaining cells use the short homologous sequence at the second DSB end to complete the repair by SDSA. BIR and SDSA products can be followed by the presence of ADE1 markers and resistance to G418 (KANMX marker). Consistent with previous assays, rad52-R70A or rad59Δ rad52-R70A derivatives nearly eliminated SDSA among the products (< 1%) while BIR increased to about 90% (Fig EV2B). Notably, overall DSB repair by BIR is more efficient in this assay when compared to the H-0 assay, as only 6–8% of the Rad52-R70A mutant cells lacking strand annealing activity failed to repair the break, resulting in chromosome loss. Thus, with much longer homology available to the invading strand, the Rad52 DNA binding domain is nearly dispensable for the completion of BIR. Click here to expand this figure. Figure EV2. Analysis of BIR efficiency in a disomic BIR system in annealing mutants Schematic of the disomic BIR assay. DSB is induced at a modified MATa locus on a truncated chromosome III, with only 46 bp homology to the right of the DSB. Strand invasion occurs within MAT sequences within the full-length chromosome III and replication continues until the end of the template chromosome. Chromosome ends are marked with ADE1, ADE3, KANMX, and HPHMX to distinguish different repair products or chromosome loss. Percentage of different repair products and chromosome loss. At least 500 colonies from YEP-Galactose plates were scored per mutant. Download figure Download PowerPoint One possible function of the DNA binding domain of Rad52 in BIR when homology is short could be related to the stability of the initial D-loop. Annealases related to Rad52, such as RecT or λ beta, were shown to promote three-strand exchange (Hall & Kolodner, 1994; Li et al, 1998), an activity that could extend the heteroduplex DNA. Alternatively, Rad52 could anneal a 3’ invading strand dissociated from its template back to the disrupted - but still RPA-bound - D-loop (Fig EV1G). Interestingly, deletion of Mph1, a D-loop disrupting helicase, slightly increased BIR efficiency in the rad52-R70A strain in the H-0 assay, although not to WT levels (Fig EV1E and F). Thus, annealing activity may counteract Mph1 by stabilizing the D-loop, at least when strand invasion is within short homologous sequences. A model presenting possible functions of Rad52’s DNA binding domain in BIR is summarized in Fig EV1G. Rad59- and Rad52-mediated annealing suppresses BIR at DSBs in regular MAT switching The above BIR/SDSA competition assays had only 46–150 bp homology on the second DSB end. To test the role of annealing in a system with longer homology, we investigated regular MAT switching, between MATa and HMLα-inc. Here, the homology on the second end is ~1,400 bp (strain H-1400) (Fig 2A). Southern blot analysis demonstrates that wild-type cells are nearly 100% efficient in repair, and neither BIR nor crossover contributes to repair. However, annealing-deficient mutants rad59Δ and particularly rad52-R70A increased BIR to ~30% and 60–70%, respectively (Fig 2B and C). As expected, viability is significantly reduced in annealing mutants due to a switch to lethal repair by BIR and inability to complete SDSA (Fig 2D). These results also demonstrate the importance of the optimal annealing mediated by Rad59 for the very fundamental event in yeast’s life cycle, mating-type switching. Figure 2. Rad52 and Rad59 suppress BIR during MAT switching Schematic of the H-1400 assay. A DSB is induced at MATa and repaired by recombination with HMLα-inc. There is ~ 1,400 bp homology between MATa and HMLα-inc sequences on the second end (green box). Representative Southern blots showing DSB repair products in WT, rad59Δ, rad52-R70A, and rad59Δ rad52-R70A cells. DNA was digested with XhoI and EcoRI and probed with a Z sequence (yellow box). A detailed map of restriction cut sites and expected size for different repair products are illustrated in Appendix Fig S1. Percentage of BIR among all repair products by 6 h (mean ± SD; n = 3). Viability of indicated strains (mean ± SD; n = 3). Graphs show analysis of repair efficiency compared to uncut parental band (left) (mean ± SD; n = 3) and repair efficiency compared to WT (right) at 6 h after DSB induction (right). See Fig 1F for details. Schematic of the H-2100 no-gap assay. A DSB is induced at MATα and repaired by recombination with HMLα-inc. There is ~ 2,100 bp homology between MATα and HMLα-inc sequences on the second end (green box). Representative Southern blots showing DSB repair products in WT, rad59Δ, and rad52-R70A. DNA was digested with XhoI and EcoRI and probed with a Z sequence (yellow box). A detailed map of restriction cut site and expected size for different repair products are illustrated in Appendix Fig S1. Percentage of BIR product among repair products by 6 h are shown (mean ± SD; n = 3). Viability of indicated strains (mean ± SD; n = 3). Graphs show analysis of repair efficiency compared to uncut parental band (left) (mean ± SD; n = 3) and repair efficiency compared to WT (right) at 6 h after DSB induction (right). See Fig 1F for details. Data information: Welch’s unpaired t-test was used to determine the P-value in all panels. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns). Download figure Download PowerPoint To eliminate the possible constraint of the gap and the non-homologous Ya tail, we tested recombination between MATα and HMLα-inc where there is no Y sequence heterology (Fig 2F). In this case, the homology on the second end is further increased to 2,100 bp (H-2100 no-gap) and is immediately next to the break. As these are MATα cells expressing Matα2, the cis-acting recombinatio
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