Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci
2016; Springer Nature; Volume: 36; Issue: 2 Linguagem: Inglês
10.15252/embj.201694628
ISSN1460-2075
AutoresYulia Vasianovich, Veronika Altmannová, Oleksii Kotenko, Matthew D. Newton, Lumír Krejčí, Svetlana Makovets,
Tópico(s)Plant Genetic and Mutation Studies
ResumoArticle17 January 2017Open Access Transparent process Unloading of homologous recombination factors is required for restoring double-stranded DNA at damage repair loci Yulia Vasianovich Yulia Vasianovich Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Veronika Altmannova Veronika Altmannova Department of Biology, Masaryk University, Brno, Czech Republic International Clinical Research Center, St. Anne's University Hospital in Brno, Brno, Czech Republic Search for more papers by this author Oleksii Kotenko Oleksii Kotenko Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Matthew D Newton Matthew D Newton Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Lumir Krejci Lumir Krejci Department of Biology, Masaryk University, Brno, Czech Republic International Clinical Research Center, St. Anne's University Hospital in Brno, Brno, Czech Republic National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic Search for more papers by this author Svetlana Makovets Corresponding Author Svetlana Makovets [email protected] orcid.org/0000-0002-0226-2093 Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Yulia Vasianovich Yulia Vasianovich Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Veronika Altmannova Veronika Altmannova Department of Biology, Masaryk University, Brno, Czech Republic International Clinical Research Center, St. Anne's University Hospital in Brno, Brno, Czech Republic Search for more papers by this author Oleksii Kotenko Oleksii Kotenko Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Matthew D Newton Matthew D Newton Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Lumir Krejci Lumir Krejci Department of Biology, Masaryk University, Brno, Czech Republic International Clinical Research Center, St. Anne's University Hospital in Brno, Brno, Czech Republic National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic Search for more papers by this author Svetlana Makovets Corresponding Author Svetlana Makovets [email protected] orcid.org/0000-0002-0226-2093 Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Author Information Yulia Vasianovich1,5, Veronika Altmannova2,3, Oleksii Kotenko1, Matthew D Newton1, Lumir Krejci2,3,4 and Svetlana Makovets *,1 1Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK 2Department of Biology, Masaryk University, Brno, Czech Republic 3International Clinical Research Center, St. Anne's University Hospital in Brno, Brno, Czech Republic 4National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic 5Present address: Department of Microbiology and Infectious Diseases, Université de Sherbrooke, Sherbrooke, QC, Canada *Corresponding author. Tel: +44 131 650 5333; E-mail: [email protected] The EMBO Journal (2017)36:213-231https://doi.org/10.15252/embj.201694628 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 Cells use homology-dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology-based mechanisms involves nuclease-dependent DNA end resection, which generates long tracts of single-stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re-synthesis of resected DNA. We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA. As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re-synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single-stranded gap and terminate further resection. Synopsis Srs2 helicase prevents DNA hyper-recombination, but is paradoxically also essential for homology-dependent double-strand break repair processes that involve extensive end resection. Srs2 facilitates completion of repair by forging ahead and preventing excessive resection, paving the way for restoration of double-stranded DNA by the replication machinery. Budding yeast Srs2 is needed for DNA re-synthesis in repair processes like de novo telomere addition, break-induced replication and single-strand annealing. Srs2-dependent Rad51 removal from single-stranded DNA promotes RPA binding necessary for PCNA loading. PCNA loading, which initiates DNA re-synthesis, may occur multiple times on longer single-strand tracts. Srs2 function at repair sites requires its translocase activity but not direct interaction with PCNA, mechanistically differentiating it from Srs2 function at replication forks. Introduction In both prokaryotes and eukaryotes, DNA double-stranded breaks (DSBs) predominantly occur as a result of broken replication forks (Vilenchik & Knudson, 2003). DSBs can also be generated due to DNA exposure to toxic chemicals or radiation as well as introduced by endogenous nucleases during developmentally programmed mechanisms such as meiosis and yeast mating type switching. DSBs are routinely repaired either by direct ligation of broken ends or by homology-dependent mechanisms such as homologous recombination (HR), break-induced replication (BIR) and single-strand annealing (SSA) (Symington et al, 2014). Alternatively, telomerase, the enzyme responsible for telomere maintenance (Greider & Blackburn, 1987), can interfere with repair by adding telomeric repeats to a DSB in a process called de novo telomere addition (Schulz & Zakian, 1994). Failure to repair DSBs results in decreased cell viability, particularly after exposure to DNA-damaging agents, increased gross chromosomal rearrangements and cancer predisposition underlying the biological significance of DNA repair mechanisms. Homology-dependent DSB repair is highly conserved in eukaryotes. In yeast Saccharomyces cerevisiae, it involves (i) initial DSB processing by MRX(Mre11-Rad50-Xrs2)/Sae2 producing a short 3′ overhang; (ii) long-range DNA resection by two redundant machineries, Dna2/Sgs1-Top3-Rmi1 and Exo1 nuclease (Mimitou & Symington, 2008; Zhu et al, 2008), which generate long tracts of ssDNA covered by the ssDNA-binding protein RPA and required for DNA damage checkpoint activation and loading of homologous recombination machinery (Zou & Elledge, 2003; Lisby et al, 2004); (iii) loading of the homologous recombination protein Rad52 followed by recruitment of Rad51 which generates a nucleoprotein filament stabilized by Rad55/57 (Symington et al, 2014). During HR and BIR, Rad52/51/55/57 promote homology search and invasion of intact donor dsDNA by the processed broken end to initiate repair (Anand et al, 2013; Symington et al, 2014). In contrast, SSA does not require DNA external to the broken chromosome as homologous sequences on either side of the break provide complementarity between the processed ends and Rad52, but not Rad51/55/57, catalyse the strand annealing (Fishman-Lobell et al, 1992; Ivanov et al, 1996). However, HR can be also toxic emphasizing the need for its tight regulation. The Srs2 helicase inhibits HR machinery by disassembling Rad51 filament and reducing DNA extension, as demonstrated in vitro (Burkovics et al, 2013; Krejci et al, 2003; Veaute et al, 2003). This function is believed to be important for repression of excessive recombination, particularly at replication forks where Srs2 is recruited and regulated through its C-terminal domain (Papouli et al, 2005; Pfander et al, 2005; Burgess et al, 2009). Loss of Srs2 results in a paradoxical phenotype. On one hand, srs2 mutants are hyper-recombinogenic (Aguilera & Klein, 1988), and on the other hand, they are deficient in DSB repair via HR and SSA (Vaze et al, 2002; Saponaro et al, 2010). Here we elucidate at the molecular level the role of Srs2 in multiple repair mechanisms involving extended DNA resection by showing that Srs2 is capable of dislodging Rad51 from ssDNA in order to promote loading of proliferating cell nuclear antigen (PCNA) and DNA replication machinery to restore dsDNA at repair loci. This function is distinct from the role of Srs2 at replication forks and essential for completion of DNA repair involving extended resection. Results Srs2 is not required for DNA damage checkpoint inactivation Cell death of srs2 mutants undergoing DSB repair is accompanied by accumulation of ssDNA and persistent activation of the DNA damage response (DDR) (Vaze et al, 2002; Yeung & Durocher, 2011). In order to distinguish between the defects of srs2 mutants in DNA repair and the recovery from DDR, we designed a system in which DSB induction led to activation of DDR, but DNA repair was not required for cells to survive DSBs (Fig 1A). In this system, one side of the break contained 81 bp of (TG1–3)n telomeric sequence which protected the centromere-proximal DNA end from resection while the other side contained either 2 or 20 kb of non-essential DNA. Only 20 kb, but not 2 kb, should be long enough to generate sufficient ssDNA post-resection to activate DDR. When the 20-kb terminal fragment becomes completely degraded, the ssDNA as a signal for checkpoint activation disappears: if cells are capable of checkpoint inactivation, they should be able to resume cycling. Figure 1. Srs2 is not required for the recovery from the DNA damage-induced arrest On the left, schematic of chr.VIIL variants, either with 2 or with 20 kb between a DSB and a telomere, used to study the effect of DNA damage checkpoint activation on cell viability in panels (B-D). Triangles represent HO sites, dashed lines represent telomeric sequences, TG81 represents 81 bp of (TG1–3)n, and grey boxes represent genes with the grey arrows above showing promoters. The diagram on the right outlines the DNA damage response (DDR) activation as the reaction of the two different strains on DSB induction by the addition of galactose (GAL). Analysis of cell cycle arrest (in G2) in response to DSB induction assayed by flow cytometry. Rad53 phosphorylation (Rad53-P) in response to DSB assayed by Western blotting. Cell survival upon DSB induction. Average ± SD (n = 4) is shown for each genotype. Data information: Strains used: NK4230, NK4231; NK4264, NK4265; NK1949; NK4268, NK4269. Download figure Download PowerPoint Activation of DDR after DSB induction was assayed by Western blotting of Rad53, the key DNA damage signalling kinase, which becomes hyper-phosphorylated in response to DNA damage. We also used FACS analysis to ask whether cells accumulate in G2 as a result of DDR activation. As expected, DSB induction in both wild-type and srs2Δ strains resulted in activation of DDR in cells with 20 kb between the break and the telomere, but not when this distance was much shorter (Fig 1B and C). However, both SRS2 and srs2Δ efficiently recovered from the cell cycle arrest as their survival was not affected by DSB induction (Fig 1D). Therefore, Srs2 is not required for the recovery from the DNA damage-induced arrest per se and the previously observed cell death of srs2Δ (Vaze et al, 2002) might come from the inability to complete DNA repair. Therefore, we next focused on the role of Srs2 in DSB repair by different mechanisms: we analysed de novo telomere addition, BIR and SSA in SRS2 and srs2Δ cells. Analysis of de novo telomere addition in SRS2 and srs2 mutant cells De novo telomere addition was assayed in SRS2 and srs2Δ using a previously described genetic test (Makovets & Blackburn, 2009) involving a single galactose-inducible DSB (Fig 2A). Because de novo telomere addition normally occurs with a very low frequency due to telomerase inhibition by Pif1 (Schulz & Zakian, 1994), the pif1-m2 background was used in the genetic assay. In srs2Δ, de novo telomere addition was reduced ~47-fold, but this effect was completely suppressed by additional deletions of RAD52, RAD51, RAD55 or RAD57 (Fig 2B). These data suggest that the presence of the HR machinery at DSBs may inhibit de novo telomere addition and that the Srs2-dependent removal of the HR proteins might reverse this inhibition. Figure 2. Srs2 is required to restore dsDNA during de novo telomere addition Schematic of the genetic de novo telomere addition assay used in (B). Cells with a galactose-inducible HO-cut are grown on YP agar with raffinose prior to plating appropriate dilutions on YPD (no DSB induction) to score cell titre, and YP with galactose to induce HO expression and DSBs. DSB repair via de novo telomere addition leads to URA3 loss and the ADH4-MNT2 locus becoming part of terminal restriction fragments containing telomeres, which can be assayed by Southern blotting. Triangle represents the HO site. Srs2 requirement in de novo telomere addition in cells with and without functional HR. All strains are pif1-m2. Strains used: NK1264; NK2375, NK2376; NK2014, NK2015; NK2451, NK2452; NK2012, NK2013; NK2457, NK2458; NK2363, NK2364; NK2469; NK2369, NK2370; NK2473–2475. Average ± SD (n = 3 or more) is shown for each genotype. Schematic of the qPCR assay used in (D) to monitor (TG1–3)n addition to DSBs. Triangle represents HO site, and blue arrows represent qPCR primers. Dynamics of (TG1–3)n addition monitored by qPCR through a time-course experiment (asynchronous populations). The y-axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalized against an internal control (ARO1 locus). Average ± SD (n = 3) is shown for each time point of each genotype. Strains used: NK3292, NK3293; NK4670, NK4671; NK4112, NK4113; NK4114, NK4115; NK3292 est2∆, NK3293 est2∆; NK4232, NK4233. Schematic of the qPCR assay coupled with PsiI digestion used in (F) to quantify ssDNA/dsDNA ratio at the de novo telomere addition locus. Numbers indicate positions of PsiI restriction sites and qPCR primer sequences relative to DSBs. Blue arrows represent qPCR primers, and dashed lines represent telomeres. Comparative analysis of ssDNA/dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time-course experiment (synchronized populations). The y-axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalized against an internal control (ARO1 locus). Average ± SD (n = 3) is shown for each time point of each genotype. Top set of error bars represents SD in relative increase of the de novo telomere-specific PCR product (as in panel D), while the lower set of error bars corresponds to quantifications of ss/dsDNA fractions. Strains used: NK3292, NK3293; NK4670, NK4671. Download figure Download PowerPoint De novo telomere addition involves (i) extension of the 3′-end as a result of addition of telomeric TG1–3 repeats by telomerase and (ii) synthesis of the complementary strand (C-strand) by the conventional replication machinery. In order to find out whether Srs2 is required at the earlier or the later step of this process, we first compared the addition of the telomeric TG1–3 repeats to the 3′-end of a break in SRS2 and srs2Δ. Cells with a galactose-inducible DSB were grown in YP + raffinose to mid-log phase and upon addition of galactose to the medium cell aliquots were taken for DNA analysis. qPCR was used to monitor addition of telomeric repeats through the time-course. One of the primers in the reaction was telomere-specific, that is consisted of AC1–3 repeats (Fig 2C), and therefore, the PCR product could be formed only after telomerase-dependent extension of the 3′-end of the break. The other primer annealed 168 bp away from the HO-cleavage site as most of the de novo telomeres in pif1-m2 are added close to the breakpoint (Schulz & Zakian, 1994). Consistent with the previously established functions of telomerase and Pif1, no addition of TG1–3 repeats to DSBs was detected in wild-type cells, where telomerase is inhibited by Pif1 (Fig 2D, dark blue), and telomerase-deficient pif1-m2 est2Δ control (Fig 2D, orange). In contrast, addition of the TG1–3 repeats in the pif1-m2 telomerase-positive yeast was readily observed (Fig 2D, light blue) and was not affected by the lack of either Srs2 (Fig 2D, pink) or Rad51/52 (Fig 2D, green). Therefore, Srs2 is not required for the telomerase-dependent addition of TG1–3 repeats to DSBs. For the completion of de novo telomere addition, the complementary C-strand needs to be synthesized all the way to the resected 5′-end. In order to monitor the conversion of the ssDNA into dsDNA, we used a previously reported approach based on digestion of qPCR template with restriction enzymes in order to differentiate between ssDNA and dsDNA (Zierhut & Diffley, 2008): if the template is single-stranded, that is synthesis of the complementary strand has not occurred, then it cannot be cleaved by a restriction enzyme. By comparing relative amounts of template DNA in parallel qPCRs with and without restriction digestion, fractions of ssDNA and dsDNA in the template DNA can be calculated as explained in Materials and Methods. Time-course experiments, where G1-arrested SRS2 and srs2Δ cells were subjected to DSB induction 1 h prior to S/G2 release into YP + galactose with nocodazole, were used to monitor the progress of de novo telomere addition both at the stage of TG1–3 repeat synthesis by telomerase and during conversion of ssDNA into dsDNA at the break. Consistent with the experiments in non-synchronized cells (Fig 2D), srs2Δ had no defect in addition of TG1–3 repeats by telomerase: during the earlier time points, the repeat addition was even more efficient in the mutants than in SRS2 (Fig 2E and F). However, when PsiI restriction enzyme was used to digest DNA templates prior to PCRs, a significant difference between SRS2 and srs2Δ in the DNA status at the breaks healed by telomerase was observed. The mutant cells consistently had higher fractions of ssDNA at multiple time points (Fig 2E and F), suggesting that conversion of ssDNA into dsDNA during de novo telomere addition was delayed in srs2Δ mutants. Thus, Srs2 is required for the conversion of the ssDNA into dsDNA after telomerase-dependent addition of TG1–3 repeats to the 3′-end and the reduced frequency of de novo telomere addition in srs2Δ in the genetic assay (Fig 2B) can be explained by the mutants' inability to restore dsDNA required in order to complete the repair. Srs2 is required for restoration of resected DNA during DSB repair by BIR Repair of DSBs via BIR involves extensive DNA resection at the break locus in order to expose ssDNA regions which are essential for the search of intact homologous sequences. The efficiency of BIR among other factors depends on the extent of homology between broken DNA ends and donor chromosomes. In order to monitor BIR by Southern blotting, we constructed a system where the usage of BIR to repair a galactose-inducible DSB was very high due to the long (~6.3 kb) homology between the broken end on chr.VIIL and the homologous sequence on chr.II (Fig 3A). In a corresponding genetic assay, ~60% of wild-type cells survived DSB induction by using BIR for repair. BIR in isogenic srs2Δ mutants was reduced to ~30% (data shown below as part of Fig 7A). Figure 3. Analysis of Srs2 requirement in BIR Schematic of the quantitative BIR assay. Modified chr.VIIL (red) and chr.IIR (blue) share a 6,272-bp homology (grey shadow) used to repair an HO-induced DSB by BIR. Black boxes indicate hybridization probes used in Southern blotting experiments to monitor re-synthesis of resected DNA (RS probes, red) and BIR (BIR probes, blue), respectively. Numbers next to the one-ended arrows indicate distances (in bp) from the homology to the distal restriction sites of the DNA fragments analysed by Southern blotting using the corresponding probes. Numbers between the restriction sites indicate the sizes of restriction fragments detected by the corresponding hybridization probes. Southern blot analysis of re-synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in (C). DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridized to the mixture of four probes (three RS probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr.V which is not involved in the repair). A representative image of one of the three repeats is shown. Re-synthesis of resected DNA on chr.VIIL in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 15.2 (RS15.2), 6.8 (RS6.8) and 2.6 (RS2.6) kb away from the homology region. Average ± SD (n = 3) is shown for each time point. Southern blot analysis of BIR-dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in (E). DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridized to the mixture of four probes (three BIR probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr.V which is not involved in the repair). A representative image of one of the three repeats is shown. C indicates control strain NK3980. BIR-dependent DNA synthesis in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 6 (BIR6), 36 (BIR36) and 77 (BIR77) kb away from the homology region. Average ± SD (n = 3) is shown for each time point. Data information: Strains used: NK4070, NK4079; NK5321, NK5322. Download figure Download PowerPoint In order to analyse progression of BIR in SRS2 and srs2Δ, a DSB on chr.VIIL was induced by expression of the HO endonuclease gene from a GAL promoter in yeast cultures arrested in G1. One hour after the HO induction, cells were released from the arrest into YP + galactose with nocodazole to prevent cell cycle progression of cells with repaired breaks. Both re-synthesis of resected DNA and BIR-dependent duplication of the chr.II fragment downstream of the homology region were monitored by quantitative Southern blotting (Fig 3B–E, respectively). Break resection is expected to convert dsDNA into ssDNA which should lead to a decrease in the hybridization signal for the corresponding restriction fragment (as ssDNA is not cut by restriction enzymes), while re-synthesis of resected DNA should restore dsDNA and the hybridization signal at the analysed locus. Analysis of DNA dynamics at three different loci on chr.VIIL, 2.6, 6.8 and 15.2 kb away from the break, showed that srs2Δ mutants had a severe defect in restoration of resected DNA (Fig 3B and C). At the 2.6 and 6.8 kb loci, the fraction of cells with dsDNA status was much lower than in the SRS2 population although the delayed restoration of dsDNA can be seen at 6 h (Fig 3C, right and middle panels). Resection may have never reached the 15.2-kb region in SRS2 (the values at all time points are close to 1), perhaps due to completion of re-synthesis before resection has reached the region (Fig 3C, left panel). At the same time, only a small fraction of srs2Δ mutants possessed dsDNA in this region by the end of the experiment (6-h time point). Therefore, Srs2 is required for re-synthesis of resected DNA during BIR. Break-induced replication in our system results in addition of ~94-kb sequence from chr.IIR to the DSB site (Fig 3A). Since ~60% of wild-type cells successfully repair DSBs by BIR, in the post-repair population the relative amount of DNA corresponding to the 94-kb sequence should increase 1.6-fold (100% on chr.IIR + 60% copied to chr.VIIL). Progression of BIR was monitored by Southern blotting using BIR6, BIR36 and BIR77 probes corresponding to DNA sequences located 6, 36 and 77 kb away from the homology region, respectively. The srs2Δ mutation resulted in slower but successful BIR-dependent DNA synthesis: like wild-type cells, srs2Δ reached 1.6-fold increase in chr.IIR sequences by the end of the time-course experiments (Fig 3D and E). Therefore, during BIR, Srs2 is predominantly required for restoring resected DNA. Srs2-dependent removal of Rad51 is necessary for efficient DNA synthesis during SSA To investigate the effect of srs2Δ on SSA, we used a genetic system where ura3-52 and URA3 were separated by ~4 kb of DNA which included KAN and a recognition site for the HO-nuclease expressed from a galactose-inducible promoter (Fig 4A). SRS2 and srs2Δ cells pre-grown on YP + raffinose agar were plated on YPD (to score total cell titre in the experiment) and YP + galactose plates for DSB induction. On galactose, upon DSB repair via SSA the vast majority of cells become Kans Ura−, as the 766-bp homology closest to the break in ura3-52 is predominantly used. The ratio between the Kans Ura− colonies grown on galactose plates and the ones on YPD was used to calculate the frequency of SSA (Fig 4B). Consistent with the previously published results (Vaze et al, 2002), srs2Δ conferred a genetic defect in SSA which was suppressed by a deletion of RAD51 (Fig 4B). Figure 4. Srs2 is required to relieve Rad51-dependent inhibition of DNA synthesis Schematic of the genetic system used to analyse inducible DSB repaired by SSA. Chr.V contains ura3-52 and URA3 (at the endogenous URA3 locus) separated by ˜4 kb of DNA containing KAN (grey box, arrow above indicates the promoter) and an HO site (triangle). Galactose-inducible expression of the HO endonuclease leads to DSB formation at the HO site. After DSB repair via SSA, the majority of cells become Kans Ura− as the 766-bp homology between URA3 and ura3-52 (grey shadows) is predominantly used. Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in (A). Average ± SD (n = 4) is shown for each genotype. Strains used: NK4691–4693; NK4805–4808; NK5081–5084; NK5085–5091. Schematic of the quantitative SSA assay used in panel (D). Grey shadow represents the annealing region of 766 bp present on both sides of a DSB. Numbers next to the one-ended arrows indicate distances (in bp) from the homology to the restriction sites, SalI and EcoRI, used to generate DNA fragment L analysed by Southern blotting. Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51. See also Fig EV3 for blot images. Average ± SD (n = 4) is shown for each time point. Strains used: NK4691–4693; NK4805–4808; NK5081–5084; NK5085–5091. Schematic for the basic DNA strand extension assay used in (F and G). Rad51 inhibits DNA synthesis of ϕX174 ssDNA substrate (0.5 nM) by Polδ. Increasing amount of Rad51 (0.03, 0.08, 0.15, 0.25, 0.5, 0.75, 1.5 μM) was incubated with the pre-loaded replication complex (RFC (17.5 nM), proliferating cell nuclear antigen (PCNA) (10 nM) and DNA) and DNA synthesis was started by the addition of Polδ (10 nM) and nucleotides containing α-32P labelled dATP. Srs2(1–910) suppresses the inhibition of DNA synthesis by Rad51. The reaction was carried out the same way as in (F) except of the increasing amount of Srs2(1–910) (5, 15, 50, 150 nM) was added to indicated reactions before the start of DNA synthesis. The relative % of DNA synthesis is indicated. Download figure Download PowerPoint Single-strand annealing involves (i) DSB processing to generate ssDNA at the regions of homology, (ii) annealing of the homologous sequences, (iii) Rad1/Rad10-dependent cleavage of the non-homologous ssDNA ends, and (iv) DNA synthesis to reconstitute DNA integrity at the repair loci (Symington et al, 2014) (Fig EV1A). To determine whether Srs2 was required at any of the first three steps, we monitored the cleavage of non-homologous ends using qPCR spanning the cleavage point but observed no significant effect of srs2Δ on the progress of SSA at this stage (Fig EV1B–E). Therefore, Srs2 loss has no effect on DSB resection (at least up to the processing of the homologous regions), annealing of the ssDNA homologies, or Rad1/Rad10-dependent cleavage of non-homologous ends and the defect of srs2Δ mutants in SSA should be attributed to a later stage of repair. Click here to expand this figure. Figure EV1. srs2Δ does not affect repair via SSA at the stage of cleavage of non-homologous DNA ends Schematic of DSB repair by SSA. Broken DNA ends are resected to generate long overhangs with 3′-ends (arrows). Once in a single-stranded form, regions of homology (thick lines) are annealed to each other. Non-homologous 3′-ends (thin lines ending with arrows) are cleaved by Rad1/Rad10, and dsDNA is restored via DNA synthesis. Sc
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