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• W2012046347 abstract "When eukaryotic chromosomes undergo double strand breaks (DSBs), several evolutionarily conserved proteins, among which the MRX complex, are recruited to the break site, leading to checkpoint activation and DNA repair. The function of the Saccharomyces cerevisiae Sae2 protein, which is known to work together with the MRX complex in meiotic DSB processing and in specific mitotic DSB repair events, is only beginning to be elucidated. Here we provide new insights into the role of Sae2 in mitotic DSB repair. We show that repair by single strand annealing of a single DSB, which is generated by the HO endonuclease between direct repeats, is defective both in the absence of Sae2 and in the presence of the hypomorphic rad50s allele altering the Rad50 subunit of MRX. Moreover, SAE2 overexpression partially suppresses the rad50s single strand annealing repair defects, suggesting that the latter might arise from defective MRX-Sae2 interactions. Finally, SAE2 deletion slows down resection of an HO-induced DSB and impairs DSB end bridging. Thus, Sae2 participates in DSB single strand annealing repair by ensuring both resection and intrachromosomal association of the broken ends. When eukaryotic chromosomes undergo double strand breaks (DSBs), several evolutionarily conserved proteins, among which the MRX complex, are recruited to the break site, leading to checkpoint activation and DNA repair. The function of the Saccharomyces cerevisiae Sae2 protein, which is known to work together with the MRX complex in meiotic DSB processing and in specific mitotic DSB repair events, is only beginning to be elucidated. Here we provide new insights into the role of Sae2 in mitotic DSB repair. We show that repair by single strand annealing of a single DSB, which is generated by the HO endonuclease between direct repeats, is defective both in the absence of Sae2 and in the presence of the hypomorphic rad50s allele altering the Rad50 subunit of MRX. Moreover, SAE2 overexpression partially suppresses the rad50s single strand annealing repair defects, suggesting that the latter might arise from defective MRX-Sae2 interactions. Finally, SAE2 deletion slows down resection of an HO-induced DSB and impairs DSB end bridging. Thus, Sae2 participates in DSB single strand annealing repair by ensuring both resection and intrachromosomal association of the broken ends. DNA double strand breaks (DSB 3The abbreviations used are: DSB, double strand break; HR, homologous recombination; SSA, single strand annealing; ssDNA, single-stranded DNA; I-SceI, intron SceI endonuclease; YEPD, yeast extract peptone dextrose; YEP+raf+gal, yeast extract peptone + raffinose + galactose; GFP, green fluorescent protein.3The abbreviations used are: DSB, double strand break; HR, homologous recombination; SSA, single strand annealing; ssDNA, single-stranded DNA; I-SceI, intron SceI endonuclease; YEPD, yeast extract peptone dextrose; YEP+raf+gal, yeast extract peptone + raffinose + galactose; GFP, green fluorescent protein.(s)) are a particularly dangerous form of DNA damage, because failure to repair these lesions can lead to loss of genetic information by deletions, duplications, translocations, and missegregation of large chromosome fragments (1.Khanna K.K. Jackson S.P. Nat. Genet. 2001; 27: 247-254Crossref PubMed Scopus (1867) Google Scholar). DSBs can arise by failures in DNA replication and by exposure to environmental factors, such as ionizing radiations and genotoxic drugs. However, they also play an important role as intermediates in meiotic and mitotic crossing over, V(D)J recombination and yeast mating type switching. When DSBs occur, many proteins are recruited to the break sites and serve both to promote a checkpoint response and to initiate DNA repair that can occur through non-homologous end joining or homologous recombination (HR) (2.Krogh B.O. Symington L.S. Annu. Rev. Genet. 2004; 38: 233-271Crossref PubMed Scopus (614) Google Scholar). Whereas non-homologous end joining implies recombination between sequences with little or no homology, HR involves exchange of genetic information between homologous DNA sequences and is the major DSB repair process in Saccharomyces cerevisiae. HR initiates with a DSB (3.Sun H. Treco D. Schultes N.P. Szostak J.W. Nature. 1989; 338: 87-90Crossref PubMed Scopus (383) Google Scholar, 4.Cao L. Alani E. Kleckner N. Cell. 1990; 61: 1089-1101Abstract Full Text PDF PubMed Scopus (531) Google Scholar), whose 5′-ends resection leaves 3′-ended single-stranded DNA (ssDNA) tails. Then, depending on the position of the homologous partner, on the initiation event and on the length of the homology region in the recombinant molecules, HR may occur by different mechanisms, including double strand break repair, synthesis-dependent strand annealing, and break-induced replication (2.Krogh B.O. Symington L.S. Annu. Rev. Genet. 2004; 38: 233-271Crossref PubMed Scopus (614) Google Scholar, 5.Paques F. Haber J.E. Micr. Mol. Biol. 1999; 63: 349-404Crossref PubMed Google Scholar). Moreover, when a DSB occurs between direct repeats, its repair is primarily achieved by a particular kind of HR named single strand annealing (SSA). SSA requires DSB resection to generate long 3′-ended single-stranded tails that can anneal with each other when resection is sufficient to uncover the duplicated sequences. Single-stranded tails are then removed by nucleases, and the resulting gaps/nicks are filled in by DNA repair synthesis and ligation, resulting in deletion of one repeat and the intervening region (6.Fishman-Lobell J. Rudin N. Haber J.E. Mol. Cell. Biol. 1992; 12: 1292-1303Crossref PubMed Scopus (280) Google Scholar). Mitotic HR involves several proteins, including Rad51, Rad52, Rad54, Rdh54/Tid1, Rad55, Rad57, Rad59, and the ssDNA binding complex replication protein A (2.Krogh B.O. Symington L.S. Annu. Rev. Genet. 2004; 38: 233-271Crossref PubMed Scopus (614) Google Scholar). Consistent with a role for Rad52 in providing the single strand annealing activity (7.Mortensen U.H. Bendixen C. Sunjevaric I. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10729-10734Crossref PubMed Scopus (379) Google Scholar, 8.Shinohara A. Shinohara M. Ohta T. Matsuda S. Ogawa T. Genes Cells. 1998; 3: 145-156Crossref PubMed Scopus (230) Google Scholar), Rad52 is essential for initiation of most HR events, including SSA, whereas Rad51, Rad54, Rad55, and Rad57 are dispensable for SSA-mediated DSB repair (9.Ivanov E.L. Sugawara N. Fishman-Lobell J. Haber J.E. Genetics. 1996; 142: 693-704Crossref PubMed Google Scholar). Different functions in DSB repair can be envisaged also for a protein complex known as MRN (Mre11-Rad50-Nbs1) in mammals and MRX (Mre11-Rad50-Xrs2) in S. cerevisiae, which has been shown to localize at meiotic and mitotic DSBs almost immediately after their formation (10.Borde V. Lin W. Novikov E. Petrini J.H. Lichten M. Nicolas A. Mol. Cell. 2004; 13: 389-401Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 11.Lisby M. Barlow J.H. Burgess R.C. Rothstein R. Cell. 2004; 118: 699-713Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). The S. cerevisiae mrx null mutants are severely impaired in meiotic recombination (4.Cao L. Alani E. Kleckner N. Cell. 1990; 61: 1089-1101Abstract Full Text PDF PubMed Scopus (531) Google Scholar, 12.Alani E. Cao L. Kleckner N. Cell. 1990; 61: 419-436Abstract Full Text PDF PubMed Scopus (476) Google Scholar) and hairpin-capped DSB repair (13.Lobachev K.S. Gordenin D.A. Resnick M.A. Cell. 2002; 108: 183-193Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar) and exhibit decreased frequencies of ionizing radiation-induced sister chromatid and interhomologue recombination (14.Bressan D.A. Baxter B.K. Petrini J.H.J. Mol. Cell. Biol. 1999; 19: 7681-7687Crossref PubMed Scopus (233) Google Scholar). Moreover, they markedly retard the kinetics of DSB repair by SSA, although they do not prevent its completion (15.Ivanov E.L. Sugawara N. White C.I. Fabre F. Haber J.E. Mol. Cell. Biol. 1994; 14: 3414-3425Crossref PubMed Scopus (203) Google Scholar). The Mre11 subunit of MRX has both exonuclease and endonuclease activities (16.Chen L. Trujillo K. Ramos W. Sung P. Tomkinson A.E. Mol. Cell. 2001; 8: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 17.Paull T.T. Gellert M. Mol. Cell. 1998; 7: 969-979Abstract Full Text Full Text PDF Scopus (691) Google Scholar, 18.Trujillo K.M. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) that likely function in DSB repair. However, whereas point mutations in the Mre11 phosphoesterase domain result in accumulation of unresected meiotic DSBs and sporulation failure in vivo (19.Moreau S. Ferguson J.R. Symington L.S. Mol. Cell. Biol. 1999; 19: 556-566Crossref PubMed Scopus (362) Google Scholar), most S. cerevisiae mre11 nuclease defective alleles cause only a mild sensitivity to ionizing radiations and do not seem to affect either HO-induced DSB resection or ionizing radiation-induced sister chromatid recombination (14.Bressan D.A. Baxter B.K. Petrini J.H.J. Mol. Cell. Biol. 1999; 19: 7681-7687Crossref PubMed Scopus (233) Google Scholar, 19.Moreau S. Ferguson J.R. Symington L.S. Mol. Cell. Biol. 1999; 19: 556-566Crossref PubMed Scopus (362) Google Scholar, 20.Lewis L.K. Storici F. Van Komen S. Calero S. Sung P. Resnick M.A. Genetics. 2004; 166: 1701-1713Crossref PubMed Scopus (68) Google Scholar, 21.Llorente B. Symington L.S. Mol. Cell. Biol. 2004; 24: 9682-9694Crossref PubMed Scopus (121) Google Scholar). Moreover, budding yeast Mre11 associates with a DSB only transiently (11.Lisby M. Barlow J.H. Burgess R.C. Rothstein R. Cell. 2004; 118: 699-713Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar), whereas resection of an HO-induced DSB can proceed for several hours until repair occurs (22.Lee S.E. Moore J.K. Holmes A. Umezu K. Kolodner R.D. Haber J.E. Cell. 1998; 94: 399-409Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). This indicates that the functions of MRX in mitotic DSB repair might be partially independent of its nuclease activity, which is instead essential in the single strand endonucleolytic removal of Spo11 from meiotic DNA ends (12.Alani E. Cao L. Kleckner N. Cell. 1990; 61: 419-436Abstract Full Text PDF PubMed Scopus (476) Google Scholar, 19.Moreau S. Ferguson J.R. Symington L.S. Mol. Cell. Biol. 1999; 19: 556-566Crossref PubMed Scopus (362) Google Scholar). Consistent with this hypothesis, the MRX complex has been shown to cooperate with Rad52 in holding the two ends of the broken chromosome together independently of its nuclease activity (23.Kaye J.A. Melo J.A. Cheung S.K. Vaze M.B. Haber J.E. Toczyski D.P. Curr. Biol. 2004; 14: 2096-2106Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 24.Lobachev K. Vitriol E. Stemple J. Resnick M.A. Bloom K. Curr. Biol. 2004; 14: 2107-2112Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Moreover, it promotes the assembly of DSB-specific cohesion domains, thus contributing to maintain association between sister chromatids during DSB repair (25.Strom L. Lindroos H.B. Shirahige K. Sjogren C. Mol. Cell. 2004; 16: 1003-1015Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 26.Unal E. Arbel-Eden A. Sattler U. Shroff R. Lichten M. Haber J.E. Koshland D. Mol. Cell. 2004; 16: 991-1002Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 27.Warren C.D. Eckley D.M. Lee M.S. Hanna J.S. Hughes A. Peyser B. Jie C. Irizarry R. Spencer F.A. Mol. Biol. Cell. 2004; 15: 1724-1735Crossref PubMed Scopus (114) Google Scholar). According to these functions, MRX structure resembles that of the SMC (structural maintenance of chromosome) proteins that are required for sister chromatid cohesion (28.Connelly J.C. Kirkham L.A. Leach D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7969-7974Crossref PubMed Scopus (193) Google Scholar). The MRX complex associates with DNA ends through the Mre11 protein and the globular domain of Rad50, with the coiled-coil arms of Rad50 extending outwards and interacting with an MRX complex on the other site of the break through Cys-Xaa-Xaa-Cys motifs (29.Stracker T.H. Theunissen J.W. Morales M. Petrini J.H. DNA Repair. 2004; 3: 845-854Crossref PubMed Scopus (228) Google Scholar). The S. cerevisiae Sae2 protein is known to work together with the MRX complex at least in meiotic DSB processing and in the repair of mitotic hairpin-capped DSBs. In fact, sae2 null mutants show meiotic recombination defects that are undistinguishable from those of hypomorphic rad50s and mre11s mutants (30.Keeney S. Kleckner N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11274-11278Crossref PubMed Scopus (164) Google Scholar, 31.McKee A.H.Z. Kleckner N. Genetics. 1997; 146: 797-816Crossref PubMed Google Scholar, 32.Nairz K. Klein F. Genes Dev. 1997; 11: 2272-2290Crossref PubMed Scopus (219) Google Scholar, 33.Prinz S. Amon A. Klein F. Genetics. 1997; 146: 781-795Crossref PubMed Google Scholar). Moreover, similarly to rad50s and mre11s mutants, sae2Δ cells are both hypersensitive to the alkylating agent methyl methanesulfonate and defective in mitotic hairpin-capped DSB repair (13.Lobachev K.S. Gordenin D.A. Resnick M.A. Cell. 2002; 108: 183-193Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 34.Baroni E. Viscardi V. Cartagena-Lirola H. Lucchini G. Longhese M.P. Mol. Cell. Biol. 2004; 24: 4151-4165Crossref PubMed Scopus (98) Google Scholar). Sae2 has neither known homologues in other species nor obvious motifs, and its function is only beginning to be elucidated. The lack of Sae2 does not affect the overall recombination rate when a DSB is induced between two inverted repeats (35.Rattray A.J. McGill C.B. Shafer B.K. Strathern J.N. Genetics. 2001; 158: 109-122Crossref PubMed Google Scholar). Nonetheless, SAE2 deletion delays Rad52 recruitment at sites of DNA damage after γ-irradiation or I-SceI-induced DSBs (11.Lisby M. Barlow J.H. Burgess R.C. Rothstein R. Cell. 2004; 118: 699-713Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar) and increases the frequency of repair events that lead to the duplication of inverted repeats (35.Rattray A.J. McGill C.B. Shafer B.K. Strathern J.N. Genetics. 2001; 158: 109-122Crossref PubMed Google Scholar) and to palindromic gene amplification (36.Rattray A.J. Shafer B.K. Neelam B. Strathern J.N. Genes Dev. 2005; 19: 1390-1399Crossref PubMed Scopus (70) Google Scholar). Because similar duplications of inverted repeats have been observed in the repair of an HO-induced DSB with only one end homologous to the donor (37.Colaiacovo M.P. Paques F. Haber J.E. Genetics. 1999; 151: 1409-1423Crossref PubMed Google Scholar), Sae2 may be involved in ensuring that both DSB ends participate in HR. To provide new insights into the Sae2 function in mitotic DSB repair, we have investigated whether the absence of Sae2 affects resection and SSA repair of a single DSB generated by the site-specific HO endonuclease. We show that Sae2 contributes to both resection and intrachromosomal association of DSB ends, thus ensuring efficient DSB repair. Yeast Strains and Media—Strains YMV80 and YMV45 were kindly provided by J. Haber (Brandeis University, Waltham, MA) and were used to disrupt the SAE2 gene and to replace RAD50 with the rad50s K81I allele (12.Alani E. Cao L. Kleckner N. Cell. 1990; 61: 419-436Abstract Full Text PDF PubMed Scopus (476) Google Scholar) or to integrate the GAL-SAE2 fusion at the URA3 locus. YMV45 and YMV80 are isogenic to YFP17 (MATΔ::hisG hmlΔ::ADE1 hmrΔ::ADE1 ade1 lys5 ura3-52 trp1 ho ade3::GAL-HO leu2::cs) except for the presence of a LEU2 fragment inserted, respectively, 4.6 or 25 kb centromere-distal to leu2::cs (38.Vaze M.B. Pellicioli A. Lee S.E. Ira G. Liberi G. Arbel-Eden A. Foiani M. Haber J.E. Mol. Cell. 2002; 10: 373-385Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). SAE2 and MRE11 disruptions were also performed in strains yJK37.2 (MATΔ hmlΔ hmrΔ can1 lys5 ade2 leu2 trp1 ura3 his3 ade3::GAL-HO VII::TRP1-HO LacI-GFP::URA3 LacO::KanR), yJK40.6 (MATΔ hmlΔ hmrΔ can1 lys5 ade2 leu2 trp1 ura3 his3 ade3::GAL-HO VII::TRP1-HO LacI-GFP::URA3 LacO::LYS5 LacO::KanR), and yJK98.9 (MATΔ hmlΔ hmrΔ can1 lys5 ade2 leu2 trp1 ura3 his3 ade3::GAL-HO VII::TRP1-HO LacI-GFP::URA3 LacO-GFP::LYS5 LacO::LEU2) (23.Kaye J.A. Melo J.A. Cheung S.K. Vaze M.B. Haber J.E. Toczyski D.P. Curr. Biol. 2004; 14: 2096-2106Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), kindly provided by D.P. Toczyski (University of California, San Francisco, CA). Strain yJK40.6 was also used to replace RAD50 with the rad50s K81I allele. SAE2 and MRE11 deletions were performed as described previously (34.Baroni E. Viscardi V. Cartagena-Lirola H. Lucchini G. Longhese M.P. Mol. Cell. Biol. 2004; 24: 4151-4165Crossref PubMed Scopus (98) Google Scholar). To generate strains carrying three copies of the GAL-SAE2 fusion integrated at the URA3 locus, the YMV80 strain was transformed with ApaI-digested plasmid pML508, carrying the entire SAE2 coding region fused to the GAL1 promoter. The YMV80 derivative SAE2-HA and GAL-SAE2-HA strains, carrying the fully functional SAE2-HA allele at the SAE2 chromosomal locus, or the GAL-SAE2-HA allele at the URA3 chromosomal locus, respectively, were constructed as described previously (34.Baroni E. Viscardi V. Cartagena-Lirola H. Lucchini G. Longhese M.P. Mol. Cell. Biol. 2004; 24: 4151-4165Crossref PubMed Scopus (98) Google Scholar). To generate rad50s mutants, YMV80 and yJK40.6 cells were transformed with MscI-digested plasmid pML533, carrying the first 666 bp of the RAD50 coding region with the K81I mutation. Accuracy of all disruptions was verified by PCR, whereas GAL-SAE2 and GAL-SAE2-HA integrations were verified by Southern blot analysis. Standard yeast genetic techniques and media were according to Ref. 39.Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar. Cells were grown in YEP medium (1% yeast extract, 2% bactopeptone, 50 mg/liter adenine) supplemented with 2% glucose (YEPD) or 2% raffinose (YEP+raf) or 2% raffinose and 2% galactose (YEP+raf+gal). Plasmids—To give rise to plasmid pML508, a fragment spanning from position +1 to position +1162 from the SAE2 translation initiation codon was amplified by PCR using yeast genomic DNA as a template and oligonucleotides PRP559 (5′-CGC GGA TCC ATA TGG TGA CTG GTG AAG AAA ATG-3′) and PRP560 (5′-AAC TGC AGC TGG TAA GTT AGG TGT CAT TTG-3′) as primers and was then cloned together with a 700-bp EcoRI-BamHI fragment containing the GAL1 promoter, into the EcoRI-PstI sites of plasmid YIplac211 (40.Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2500) Google Scholar). To give rise to plasmid pML533, a 1051-bp fragment containing the promoter and the first 666 bp of the coding region of the rad50s K81I allele was amplified by PCR using oligonucleotides PRP706 (5′-CGG AAT TCC CGA TAG TAC TTC CAC TTA CAA TAC-3′) and PRP707 (5′-CGG AAT TCC ATT GCT TTC GAT CTG TCT TTA TCC-3′) as primers and yeast genomic DNA from MJL1699 strain (kindly provided by N. Kleckner, Harvard University, Cambridge, MA) as a template and was then cloned into the EcoRI site of plasmid YIplac128 (40.Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2500) Google Scholar). The presence of the mutation was verified by nucleotide sequence analysis. Other Techniques—Synchronization experiments, protein extract preparation, and Western blot analysis were performed as previously described (41.Clerici M. Baldo V. Mantiero D. Lottersberger F. Lucchini G. Longhese M.P. Mol. Cell. Biol. 2004; 24: 10126-10144Crossref PubMed Scopus (37) Google Scholar). DSB formation and repair were detected by Southern blot analysis using an Asp718-SalI fragment containing part of the LEU2 gene as a probe (38.Vaze M.B. Pellicioli A. Lee S.E. Ira G. Liberi G. Arbel-Eden A. Foiani M. Haber J.E. Mol. Cell. 2002; 10: 373-385Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). DSB end resection at the LEU2 locus was analyzed on alkaline-agarose gels as described in Ref. 42.Shroff R. Arbel-Eden A. Pilch D. Ira G. Bonner W.M. Petrini J.H. Haber J.E. Lichten M. Curr. Biol. 2004; 14: 1703-1711Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, using a single-stranded probe complementary to the unresected DSB strand. This probe was obtained by in vitro transcription using Promega Riboprobe System-T7 and plasmid pML514 as a template. Plasmid pML514 was obtained by cloning the KpnI-ClaI fragment containing part of the LEU2 gene in the KpnI-ClaI sites of pGEM-7Zf plasmid (purchased from Promega). The Lack of Sae2 Impairs DSB Repair by SSA—We examined the effects caused by lack or excess of Sae2 on SSA repair by using YMV80 derivative strains (38.Vaze M.B. Pellicioli A. Lee S.E. Ira G. Liberi G. Arbel-Eden A. Foiani M. Haber J.E. Mol. Cell. 2002; 10: 373-385Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), carrying either a deletion of SAE2 or a GAL1-SAE2 fusion integrated at the URA3 locus. In these strains, a single DSB can be generated by expressing the site-specific HO endonuclease gene from a galactose-inducible GAL1 promoter. The normal HO recognition sites at MAT, HML, and HMR loci are deleted, whereas an HO target sequence on chromosome III is flanked on either side by homologous sequence repeats, consisting of the 3′-end of the LEU2 gene. One of these repeats is adjacent to the HO cut site (leu2::cs), whereas the other repeat (his4::leu2) is located 25 kb centromere distal from the break site (Ref. 38.Vaze M.B. Pellicioli A. Lee S.E. Ira G. Liberi G. Arbel-Eden A. Foiani M. Haber J.E. Mol. Cell. 2002; 10: 373-385Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar; see also Fig. 1A). Because of the presence of homologous sequence repeats on either side of the HO cut site, the DSB in the YMV80 derivative strains has been reported to be mainly repaired by a Rad52-dependent SSA pathway (38.Vaze M.B. Pellicioli A. Lee S.E. Ira G. Liberi G. Arbel-Eden A. Foiani M. Haber J.E. Mol. Cell. 2002; 10: 373-385Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). In fact, the ends of the DSB are degraded by 5′-3′-exonucleases until complementary sequences flanking the break are exposed and can be annealed. Subsequent ligation of the two chromosomal fragments repairs the break, concomitantly abolishing the HO recognition sequence and one of the repeats (38.Vaze M.B. Pellicioli A. Lee S.E. Ira G. Liberi G. Arbel-Eden A. Foiani M. Haber J.E. Mol. Cell. 2002; 10: 373-385Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Because the ability to repair the HO-induced DSB is influenced by the cell cycle stages (43.Ira G. Pellicioli A. Balijja A. Wang X. Fiorani S. Carotenuto W. Liberi G. Bressan D. Wan L. Hollingsworth N.M. Haber J.E. Foiani M. Nature. 2004; 431: 1011-1017Crossref PubMed Scopus (571) Google Scholar), we arrested all cell cultures in mitosis and kept them blocked by nocodazole treatment during break induction by galactose addition. We then monitored the kinetics of HO-cut formation and its subsequent repair by Southern blot analysis. As depicted in Fig. 1A, hybridization with a LEU2 probe of KpnI-digested YMV80 genomic DNA run on a native agarose gel reveals 8- and 6-kb DNA fragments in the absence of HO cut. HO-induced DSB formation results in decrease of the amount of 6-kb species and concomitant appearance of 2.5-kb fragments. The break can be eventually repaired by SSA, leading to a final product of 3.5-kb and disappearance of both the 2.5- and 8-kb fragments. As shown in Fig. 1B, HO-cut induction was rapid and efficient in all cell cultures, because the leu2::cs sequence was cut in more than 90% of cells within 30 min after galactose addition. Then, the 3.5-kb SSA repair products started to accumulate in both wild type and SAE2 overexpressing cells ∼5 h after HO induction, concomitantly with the decrease of both the 2.5- and 8-kb fragments. SSA products were instead detectable only at very low levels even 10 h after HO induction in sae2Δ cells (Fig. 1B). Consistent with defective DSB repair by SSA in the absence of Sae2, only 10% of sae2Δ cells were able to form colonies on galactose-containing plates (HO expression on), whereas cell viability was unaffected in wild type and GAL-SAE2 cells under the same conditions (Fig. 1C). Thus, Sae2 is required for efficient repair of the HO-induced DSB by SSA, which appears to be unaffected by SAE2 overexpression, indicating that increasing Sae2 levels (Fig. 1D) is not sufficient to accelerate this process in wild type cells. The lack of Sae2 severely reduced SSA-dependent recombination events also in YMV45 derivative strains (Fig. 2), where SSA repair of HO-induced DSBs occurs very rapidly (38.Vaze M.B. Pellicioli A. Lee S.E. Ira G. Liberi G. Arbel-Eden A. Foiani M. Haber J.E. Mol. Cell. 2002; 10: 373-385Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). In fact, the distal leu2 sequence in these strains is located only 4.6 kb away from the HO recognition site at leu2::cs (Ref. 38.Vaze M.B. Pellicioli A. Lee S.E. Ira G. Liberi G. Arbel-Eden A. Foiani M. Haber J.E. Mol. Cell. 2002; 10: 373-385Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, see also Fig. 2A). When cell cultures of these strains were arrested in mitosis and kept blocked by nocodazole treatment during break induction, SSA products accumulated in wild type cells already 90 min after HO induction (Fig. 2B). Moreover, they became detectable in sae2Δ cells ∼150 min after galactose addition (Fig. 2B), but their amount was much lower than in wild type, thus confirming a critical role for Sae2 in SSA repair. Consistent with the faster DSB repair by SSA in YMV45 sae2Δ cells compared with YMV80 sae2Δ cells, 50% of YMV45 sae2Δ cells were able to form colonies on galactose-containing plates (Fig. 2C). SAE2 Overexpression Partially Suppresses the SSA Defects of rad50s Mutants—In contrast to the mrx null alleles, which are unable to generate meiotic DSBs, sae2Δ cells display the same meiotic recombination defects as the hypomorphic rad50s and mre11s alleles (30.Keeney S. Kleckner N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11274-11278Crossref PubMed Scopus (164) Google Scholar, 31.McKee A.H.Z. Kleckner N. Genetics. 1997; 146: 797-816Crossref PubMed Google Scholar, 32.Nairz K. Klein F. Genes Dev. 1997; 11: 2272-2290Crossref PubMed Scopus (219) Google Scholar, 33.Prinz S. Amon A. Klein F. Genetics. 1997; 146: 781-795Crossref PubMed Google Scholar), allowing to hypothesize that the Rad50s and Mre11s mutant proteins might specifically impair MRX-Sae2 interaction. We then asked whether rad50s mutant cells were defective in SSA. After construction of a rad50s K81I mutant (12.Alani E. Cao L. Kleckner N. Cell. 1990; 61: 419-436Abstract Full Text PDF PubMed Scopus (476) Google Scholar) in the YMV80 background, cell cultures were arrested in mitosis and kept blocked by nocodazole treatment during break induction by galactose addition. Southern blot analysis was then used to follow the kinetics of DSB repair by SSA. As shown in Fig. 3A, SSA products were barely detectable in rad50s cells even 8 h after HO induction, similarly to what observed in isogenic sae2Δ cells (Fig. 1B). Accordingly, rad50s cell survival on galactose containing plates was reduced to the same extent as in sae2Δ cells (Fig. 3B). If the rad50s SSA defects were because of altered MRX ability to interact with Sae2, Sae2 overproduction might be expected to suppress it. This was indeed the case, because SSA products in GAL-SAE2 rad50s cells treated as above became detectable and started to accumulate, although to a lower extent than in wild type, ∼5 h after HO induction (Fig. 3A). In agreement with partial suppression of rad50s SSA defects by SAE2 overexpression, GAL-SAE2 rad50s cells showed increased cell survival after HO induction on galactose-containing plates compared with rad50s cells (Fig. 3B). The Lack of Sae2 Slows Down Resection of an HO-induced DSB—Because the HO break can undergo repair by SSA only once 5′-3′-resection has uncovered both direct repeats, allowing their subsequent annealing and ligation, SSA repair defects in sae2Δ cells might be because of impairments in the generation of 3′-ended ssDNA. We therefore used denaturing gel electrophoresis and Southern blot analysis with a single-stranded LEU2 probe to monitor both HO cut at leu2::cs and the extent of resection, as depicted in Fig. 4A. When these analyses were performed on the same cell cultures shown in Fig. 1, initiation of DSB resection turned out to be delayed in YMV80 sae2Δ cells (Fig. 4B). In fact, 3′-ended resection products (named r1-r6) could be detected in these cells ∼1 h later than in isogenic wild type cells. The sae2Δ DSB resection defects seemed to only partially account for the reduced SSA efficiency. In fact, the amount of SSA products was very low for at least 10 h after HO induction in YMV80 sae2Δ cells (Fig. 1B), whereas the 3′-ended ssDNA products were detectable in the same cells already 2-3 h after galactose addition and remained stable for at least 10 h (Fig. 4B). Conversely, the amount of resection products decreased in the isogenic wild type cells (Fig. 4B) concomitantly with the appearance of SSA products (Fig. 1B). Moreover, DSB resection in the sae2Δ YMV80 derivative strain seemed to proceed for at least 25 kb and to uncover the homologous LEU2 sequence (his4::leu2) located 25 kb away from the HO-cut site. In fact, the amount of the 8-kb his4::leu2 DNA fragment started to decrease ∼6-7 h after HO induction in both wild type and sae2Δ YM" @default.
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• W2012046347 title "The Saccharomyces cerevisiae Sae2 Protein Promotes Resection and Bridging of Double Strand Break Ends" @default.
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