DNA Structure-specific Nuclease Activities in theSaccharomyces cerevisiae Rad50·Mre11 Complex

Abstract

Saccharomyces cerevisiae RAD50 and MRE11 genes are required for the nucleolytic processing of DNA double-strand breaks. We have overexpressed Rad50 and Mre11 in yeast cells and purified them to near homogeneity. Consistent with the genetic data, we show that the purified Rad50 and Mre11 proteins form a stable complex. In the Rad50·Mre11 complex, the protein components exist in equimolar amounts. Mre11 has a 3′ to 5′ exonuclease activity that results in the release of mononucleotides. The addition of Rad50 does not significantly alter the exonucleolytic function of Mre11. Using homopolymeric oligonucleotide-based substrates, we show that the exonuclease activity of Mre11 and Rad50·Mre11 is enhanced for substrates with duplex DNA ends. We have examined the endonucleolytic function of Mre11 on defined, radiolabeled hairpin structures that also contain 3′ and 5′ single-stranded DNA overhangs. Mre11 is capable of cleaving hairpins and the 3′ single-stranded DNA tail. These endonuclease activities of Mre11 are enhanced markedly by Rad50 but only in the presence of ATP. Based on these results, we speculate that the Mre11 nuclease complex may mediate the nucleolytic digestion of the 5′ strand at secondary structures formed upon DNA strand separation. Saccharomyces cerevisiae RAD50 and MRE11 genes are required for the nucleolytic processing of DNA double-strand breaks. We have overexpressed Rad50 and Mre11 in yeast cells and purified them to near homogeneity. Consistent with the genetic data, we show that the purified Rad50 and Mre11 proteins form a stable complex. In the Rad50·Mre11 complex, the protein components exist in equimolar amounts. Mre11 has a 3′ to 5′ exonuclease activity that results in the release of mononucleotides. The addition of Rad50 does not significantly alter the exonucleolytic function of Mre11. Using homopolymeric oligonucleotide-based substrates, we show that the exonuclease activity of Mre11 and Rad50·Mre11 is enhanced for substrates with duplex DNA ends. We have examined the endonucleolytic function of Mre11 on defined, radiolabeled hairpin structures that also contain 3′ and 5′ single-stranded DNA overhangs. Mre11 is capable of cleaving hairpins and the 3′ single-stranded DNA tail. These endonuclease activities of Mre11 are enhanced markedly by Rad50 but only in the presence of ATP. Based on these results, we speculate that the Mre11 nuclease complex may mediate the nucleolytic digestion of the 5′ strand at secondary structures formed upon DNA strand separation. double-strand break 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol adenosine 5′-O-(thiotriphosphate) single-stranded DNA double-stranded DNA polyacrylamide gel electrophoresis Tris borate EDTA DSBs are induced by ionizing radiation and are also formed during initiating events in various modes of homologous recombination (1Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar). Genetic studies in Saccharomyces cerevisiae have been instrumental in the discovery of genes required for recombination and DSB1 repair through homologous recombination. These genes, RAD50,MRE11, XRS2, RAD51, RAD52,RAD54, RAD55, RAD57, RAD59, and RDH54/TID1, are collectively referred to as theRAD52 epistasis group (2Sung P. Trujillo K.M. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (159) Google Scholar). A number of studies in S. cerevisiae indicate that DSBs in homologous recombination events are processed in a nucleolytic fashion to generate an intermediate with overhanging 3′ ssDNA tails. The ssDNA tails are bound by Rad51 and other recombination factors, which function in concert to locate regions of homology on a corresponding DNA duplex (a homologous chromosome or sister chromatid) and form heteroduplex DNA joints (1Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar, 2Sung P. Trujillo K.M. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (159) Google Scholar). RAD50, MRE11, and XRS2 are involved in the nucleolytic processing of DSBs. Analysis of the Rad50 and Mre11 sequences reveals homology of these two proteins to Escherichia coli SbcC and SbcD, respectively (3Sharples G.J. Leach D.F.R. Mol. Microbiol. 1995; 17: 1215-1217Crossref PubMed Scopus (195) Google Scholar), which combine to form a complex that exhibits both exo- and endonuclease activities, including the capacity to cleave hairpin structures (4Connelly J.C. Kirkham L.A. Leach D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7969-7974Crossref PubMed Scopus (199) Google Scholar). Mre11 from human and yeast possesses exo- and endonuclease activities (5Furuse M. Nagase Y. Tsubouchi H. Murakami-Murofushi K. Shibata T. Ohta K. EMBO J. 1998; 17: 6412-6425Crossref PubMed Scopus (222) Google Scholar, 6Moreau S. Ferguson J.R. Symington L.S. Mol. Cell. Biol. 1999; 19: 556-566Crossref PubMed Scopus (364) Google Scholar, 7Usui T. Ohta T. Oshiumi H. Tomizawa J. Ogawa H. Ogawa T. Cell. 1998; 95: 705-716Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 8Trujillo K.M. Yuan S.S.F. Lee E.Y-H.P. Sung P. J. Biol. Chem. 1998; 273: 21447-21450Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 9Paull T.T. Gellert M. Mol. Cell. 1998; 1: 969-979Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Interestingly, overexpression of Rad50 and Mre11 can allow DNA synthesis to efficiently progress through DNA sequences with a propensity to form secondary structures, suggesting that the Mre11 complex in yeast might also have the ability to cleave such DNA structures (10Richard G.F. Goellner G.M. McMurray C.T. Haber J.E. EMBO J. 2000; 19: 2381-2390Crossref PubMed Scopus (118) Google Scholar). As evidenced by co-immunoprecipitation and two-hybrid experiments, Rad50, Mre11, and Xrs2 are associated in a complex (7Usui T. Ohta T. Oshiumi H. Tomizawa J. Ogawa H. Ogawa T. Cell. 1998; 95: 705-716Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 11Johzuka K. Ogawa H. Genetics. 1995; 139: 1521-1532Crossref PubMed Google Scholar). Likewise, the human counterparts of these proteins, human Rad50, Mre11, and NBS1 (the Xrs2 equivalent) have also been purified from human cells as an endogenous complex (8Trujillo K.M. Yuan S.S.F. Lee E.Y-H.P. Sung P. J. Biol. Chem. 1998; 273: 21447-21450Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Remarkably, genetic evidence in S. cerevisiae implicates RAD50, MRE11, andXRS2 in other cellular functions including the formation of DSBs at meiotic recombination hotspots (1Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar), non-homologous DNA end-joining, and the maintenance of telomere length (1Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar, 12Boulton S.J. Jackson S.P. EMBO J. 1998; 17: 1819-1828Crossref PubMed Scopus (556) Google Scholar). More recently, Rad50 and Mre11 have been implicated in the adaptation to a Rad9/Rad17-mediated G2/M checkpoint after the introduction of an unrepairable DSB (13Lee 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 (651) Google Scholar). The multifunctional nature of the Rad50, Mre11, and Xrs2 trio emphasizes the importance for the biochemical characterization of these proteins, singly and in combination, for activities germane for their biological functions. Here, we purify Rad50 and Mre11, reconstitute the Rad50·Mre11 complex, and describe the endo- and exonucleolytic activities of these factors. We speculate on how the DNA structure-specific endonuclease activity of Rad50·Mre11 could be utilized to create ssDNA tails that facilitate subsequent steps in recombination, including the formation of dual Holliday junctions. TheRAD50 and MRE11 genes were introduced into the vector pPM231 containing the galactose-inducible hybridGAL-PGK promoter. The resulting plasmids, pR50.1 (2 μm, GAL-PGK-RAD50, LEU-2d) and pM11.1 (2 μm, GAL-PGK-MRE11, LEU-2d), were introduced into the protease-deficient strain BJ5464 (MATα ura3–52 trp1 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1). Cultures were grown overnight to the stationary phase in synthetic medium lacking leucine and diluted 8-fold into leucine dropout synthetic media with 3% glycerol, 3% lactic acid, and 2% galactose. After 24 h of growth at 30 °C, cells were harvested by centrifugation. All the steps were carried out at 0 to 4 °C. Typically, cells (50 g) expressing Rad50 were resuspended in 75 ml of cell breakage buffer (50 mm Tris-HCl, pH 7.5, 10% sucrose, 10 mm EDTA, 1.2 m KCl, 1.0 mm dithiothreitol, and 0.01% Nonidet P-40) with protease inhibitors (5.0 μg/ml each of aprotinin, leupeptin, chymostatin, pepstatin A, and 1 mm each phenylmethylsulfonyl fluoride and benzamidine) and lysed in a French press (SLM Aminco) at 20,000 p.s.i. The soluble fraction (Fraction I) was collected by ultracentrifugation (100,000 × g, 90 min) and treated with ammonium sulfate at 0.28 g/ml. The precipitate was collected by centrifugation (17,000 × g, 25 min), redissolved in 100 ml of K buffer (20 mm KH2PO4, pH 7.4, 10% glycerol, 0.5 mm EDTA, 0.01% Nonidet P-40, and 1.0 mm dithiothreitol) with protease inhibitors, and dialyzed against K buffer + 100 mm KCl. The dialysate (Fraction II) was applied to a 20-ml Q-Sepharose column, which was developed over a 200-ml gradient from 100 to 540 mm KCl, collecting 5.0-ml fractions. The peak fractions (about 350 mm KCl, Fraction III), as identified by Coomassie Blue staining of SDS-PAGE gels, were pooled and applied directly onto an 8.0-ml Affi-Gel Blue (Bio-Rad) column, which was developed with an 80-ml gradient from 100 to 1800 mm KCl, collecting 2.0-ml fractions. Peak elution occurred at about 1400 mm KCl, and these fractions were pooled (Fraction IV), dialyzed against K buffer + 100 mm KCl, and fractionated in a 6.0-ml macro-hydroxyapatite (Bio-Rad) column with a 50-ml gradient from 0 to 240 mm KH2PO4 in K buffer + 100 mm KCl, collecting 1.0-ml fractions. The peak fractions (∼175 mm KH2PO4, Fraction V) were pooled, concentrated in a Centricon 30 concentrator to 0.7 ml, and subjected to gel filtration on a 35-ml Sepharose 6B column in K buffer + 100 mm KCl. The peak of Rad50 (eluted at 16–22 ml, Fraction VI) was further purified in a 1.0-ml Mono Q column using a 40-ml gradient from 100 to 370 mm KCl, collecting 1.0-ml fractions. Rad50 (eluted at about 170 mm KCl, Fraction VII) was concentrated in a Centricon 30 microconcentrator to a final volume of 1.0 ml at 10 mg/ml. Rad50 was stored in small aliquots at −80 °C. The Rad50 protein concentration was determined by densitometric scanning of a 7.5% SDS-PAGE gel containing multiple loadings of purified Rad50 against known amounts of bovine serum albumin run on the same gel. All the steps were carried out at 0–4 °C. Typically, extract (Fraction I) was prepared from yeast cells (100 g) expressing Mre11 and subjected to ammonium sulfate precipitation at 0.28 g/ml. The resulting precipitate was dissolved in 130 ml of K buffer with protease inhibitors and dialyzed against K buffer + 100 mm KCl. The dialysate (Fraction II) was applied to a 25-ml Q-Sepharose column developed with a 250-ml gradient of 100–650 mm KCl, collecting 5.0-ml fractions. The peak fractions of Mre11 (∼300 mm KCl, Fraction III), as identified by Coomassie Blue staining of SDS-PAGE gels, were combined, bound to a 6.0-ml macro-hydroxyapatite column, and eluted with a gradient of 0–270 mm KH2PO4 in K buffer + 100 mm KCl. The Mre11 peak fractions from the macro-hydroxyapatite step (∼140 mmKH2PO4) were pooled (Fraction IV), concentrated using a Centricon 30 concentrator to ∼4.0 ml, and subjected to gel filtration on a 200-ml Sephacryl 300 column in K buffer + 100 mm KCl. Mre11-containing fractions (eluted at 90–120 ml, Fraction V) were loaded onto a 1.0-ml Mono S column, which was developed with a 40-ml KCl gradient from 100 to 630 mm KCl, with Mre11 eluting at about 300 mm KCl. The peak fractions (Fraction VI) were concentrated in a Centricon 30 microconcentrator to a final volume of 2.0 ml at 10 mg/ml. Purified Mre11 was stored at −80 °C in small aliquots. The Mre11 protein concentration was determined by densitometric scanning of a 7.5% SDS-PAGE gel containing multiple loadings of purified Mre11 against known amounts of bovine serum albumin run on the same gel. Fraction Rad50 was with Fraction Mre11 in ml and on for h to allow for complex The Rad50·Mre11 complex was from Mre11 by gel filtration on a 200-ml Sepharose 6B column in K buffer + 100 mm KCl. were collected over a 200-ml with the Rad50·Mre11 complex eluting at The Rad50·Mre11 complex was further purified on a 1.0-ml Mono S column with a 40-ml gradient mm with the complex eluting at mm KCl. The peak fractions were pooled and concentrated to a final volume of 1.0 ml at 10 mg/ml. were from T.T. Gellert M. Mol. Cell. 1998; 1: 969-979Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar), a the T.T. Gellert M. Mol. Cell. 1998; 1: 969-979Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar), also a was the of was to for the of the 3′ of and of A the and the These were purified on polyacrylamide containing m and run at °C in 100 mm Tris pH and 2.0 mm EDTA The were from gel dialyzed against and concentrated in Centricon 10 The single-stranded as was from DNA hairpin substrates were prepared by and at the 3′ with and The were at °C for 10 and to to to The was further purified on a 10% polyacrylamide gel in at 4 °C and by The 5′ duplex substrates were by radiolabeled at the 5′ with and to at °C in 30 mm Tris pH with mm KCl for 10 which was to to were determined by on a 10% polyacrylamide gel in against known amounts of DNA and by The 3′ duplex substrates were by the of to and subsequent of the resulting with the and The was by by gel filtration in (Bio-Rad) to the and were at the 3′ with and or that the was at the 3′ with an A, and with a T. these a of to was to the that multiple were to a The was by to DNA substrates were with Mre11 or Rad50·Mre11 complex in in buffer mm pH with mm KCl, mm μg/ml bovine serum and 1.0 mm or were also After at or 4 °C the was by volume of 3% and with K at °C for 10 oligonucleotide-based nuclease volume of with was to each which was for polyacrylamide containing m and in 100 mm Tris pH and 2.0 mm EDTA After the were in 10% acid, 10% and subjected to and of the exonuclease were loaded onto T. which were developed in 1.0 and m were by and by SDS-PAGE of from yeast cells the Rad50 and the vector the Rad50 protein The of the overexpressed Rad50 protein 1 was in with the for protein Res. PubMed Scopus Google Scholar). we have that Rad50 in extract has the same gel as the overexpressed Rad50 was purified 1 to near 1 Likewise, of from yeast cells the Mre11 pPM231 the overexpression of Mre11 1 The gel of for Mre11 was the of for protein K. Ogawa H. Genetics. 1995; 139: 1521-1532Crossref PubMed Google Scholar). we have that the overexpressed Mre11 the same gel as Mre11 from Mre11 was also purified to near purified Rad50 and Mre11 were on and applied to a Sepharose 6B gel filtration column, the Mre11 was to with Rad50 as a suggesting that Rad50 and Mre11 formed a stable complex. Interestingly, Rad50 not to a Mono S column, was efficiently after with Mre11, which to Mono evidence that these proteins form a stable complex. biochemical the Rad50·Mre11 complex was and purified to near as and to of equimolar amounts of the two proteins 1 the addition of on complex the exonuclease activity of Mre11 and the Rad50·Mre11 complex, the duplex and the duplex substrates were from the of the the and the the same DNA the the was carried out at 4 °C. of the by Mre11 or the Rad50·Mre11 complex as a function of a of radiolabeled of digestion of the a that at the gel These results that the exonuclease activity of Mre11 is 3′ to 5′ in Likewise, the Rad50·Mre11 complex a 3′ to 5′ exonuclease In to human Rad50, which the exonuclease activity of human Mre11 T.T. Gellert M. Mol. Cell. 1998; 1: 969-979Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar), yeast Rad50 to have on the exonuclease activity of Mre11. The exonuclease by the digestion of the by Mre11 and Rad50·Mre11 was a as determined by to the of the by the 3′ to 5′ from coli by and on polyacrylamide exonuclease activity of Mre11 and the Rad50·Mre11 complex is on The exonuclease activity of Mre11 and Rad50·Mre11 on the 3′ was in the presence of or as were carried out as in for and were by and the by exonuclease were run as The exonuclease activity of Mre11 and Rad50·Mre11 required which could not be by or As the exonuclease activity of Mre11 was by or Interestingly, was not a for the Rad50·Mre11 exonuclease A and of the was from the duplex by Rad50·Mre11 with or in only was in the presence of A These results that the form of Rad50·Mre11 is results that Rad50 and the Rad50·Mre11 complex only a of activity with of and at °C, The exonuclease activity of Mre11 and Rad50·Mre11 was from pH to and was pH As Rad50 was of exonuclease activity We examined Mre11 and Rad50·Mre11 also on was at the 3′ with an A or as of these substrates with Rad50·Mre11 or Mre11 in the release of as for the was in with the of (7Usui T. Ohta T. Oshiumi H. Tomizawa J. Ogawa H. Ogawa T. Cell. 1998; 95: 705-716Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). we also to the that of the ssDNA in to sequences the or might generate a duplex for the Rad50·Mre11 exonuclease function to we the homopolymeric and of a run of or A at the 3′ with a or A substrates be of secondary As in Rad50·Mre11 ability to these the by the radiolabeled with the was by Rad50·Mre11 the We to the that the is a of of which have ssDNA or have the radiolabeled in the from results were using Mre11 not and with exonuclease which is a known 3′ to 5′ exonuclease the results that the exonuclease activity of Mre11 and Rad50·Mre11 has a for duplex DNA and that digestion of the as was enhanced by events that duplex DNA ends. Mre11 (5Furuse M. Nagase Y. Tsubouchi H. Murakami-Murofushi K. Shibata T. Ohta K. EMBO J. 1998; 17: 6412-6425Crossref PubMed Scopus (222) Google Scholar, 6Moreau S. Ferguson J.R. Symington L.S. Mol. Cell. Biol. 1999; 19: 556-566Crossref PubMed Scopus (364) Google Scholar, 7Usui T. Ohta T. Oshiumi H. Tomizawa J. Ogawa H. Ogawa T. Cell. 1998; 95: 705-716Abstract Full Text Full Text PDF PubMed Scopus (308) Google and Rad50·Mre11 not were capable of ssDNA to of gel the of an The that Mre11 Rad50·Mre11 a homopolymeric that might be on secondary structures the ssDNA was of to the endonucleolytic function on hairpin substrates that structures in the ssDNA in endo- exonuclease activities on such substrates were by the of a homopolymeric at the of the by that such were to exonucleolytic Interestingly, both Mre11 and Rad50·Mre11 were capable of resulting in the of two endonucleolytic A and A was the of an at the of the hairpin to the as determined by of on gels, the of the the same of a hairpin or a hairpin with a homopolymeric was from at the of the duplex with the 3′ ssDNA to the and min) for these with the °C and min) to the exonuclease activity of the homopolymeric substrates in a of was We the of was for the structure-specific endonucleolytic function of Mre11 and In the of Rad50·Mre11 was to Mre11 with to endonuclease activity and in the presence of the endonuclease activity of the Rad50·Mre11 complex was that of Mre11 was and only of was to A and by Rad50·Mre11 after in the of was into these was not the endonuclease activity of Rad50·Mre11 and have on the endonucleolytic function of Mre11. As the structure-specific endonuclease activities a for as Mre11 and Rad50·Mre11 exonuclease activity with a 3′ to 5′ that Mre11 Rad50·Mre11 are significantly by for exonuclease the activity of Rad50·Mre11 is by Interestingly, has been to the exonuclease activity of human Mre11 T.T. Gellert M. Mol. Cell. 1998; 1: 969-979Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar), we that S. cerevisiae Rad50 does not to significantly the exonuclease activity of S. cerevisiae Mre11. We also that Mre11 with or Rad50 has a for duplex DNA ends. The ability of Mre11 to ssDNA substrates as by T. Ohta T. Oshiumi H. Tomizawa J. Ogawa H. Ogawa T. Cell. 1998; 95: 705-716Abstract Full Text Full Text PDF PubMed Scopus (308) Google was enhanced by the formation of duplex the Based on that the exonuclease activity of Mre11 was by homopolymeric ssDNA we hairpin structures with to allow to the endonuclease Using we were to two endonucleolytic activities hairpin of the endonucleolytic was formed upon at the of a hairpin to the 3′ The endonucleolytic was formed upon at the the duplex DNA and the 3′ ssDNA Mre11 was capable of both Rad50 these activities but was required for the of the endonucleolytic activities of only the Rad50·Mre11 complex. DNA structure-specific endonuclease activities were by and Gellert T.T. Gellert M. 1999; PubMed Scopus Google for the human Mre11 but results to two how these endonucleolytic activities in the yeast and human counterparts are the ability to a DNA hairpin and a 3′ ssDNA for is on both the human Rad50 and NBS1 proteins T.T. Gellert M. 1999; PubMed Scopus Google Scholar), S. cerevisiae Rad50 is to these activities in yeast Mre11, that Xrs2 may further the endonucleolytic function of the of a 3′ ssDNA but not a DNA hairpin by the human complex the yeast Rad50·Mre11 complex for the of both structure-specific from a number of genetic studies in S. cerevisiae indicate that during DSB the DNA that contain the 5′ are (1Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar). is that the Mre11 exonuclease which has a 3′ to 5′ be for the of the 5′ DNA human yeast Mre11 also possesses the ability to DNA hairpin structures as as 3′ ssDNA tails that a duplex The nature of these endonucleolytic activities that are germane for the processing of DNA DSBs. We speculate that these DNA structure-specific activities may be in with a DNA to DNA double-strand breaks. The of the DNA by the complex and of a DNA to of the two DNA 3′ and 5′ single-stranded A, We also that may be by a ssDNA by the 3′ to 5′ exonuclease activity of the complex or through the capacity of Rad50 to duplex DNA T.T. Gellert M. 1999; PubMed Scopus Google Scholar). As in A, the endonucleolytic function of the complex may and secondary structures in the DNA that the 5′ of the DNA break A, the of DSB processing in during which the of DSBs are upon by the of the protein complex R.D. P. Rev. 1995; PubMed Google Scholar). The of such a of processing in create not only a 3′ ssDNA but also a 5′ ssDNA as A, that Rad51 can efficiently both 3′ and 5′ ssDNA tails for DNA formation Sung P. EMBO J. 2000; 19: PubMed Scopus Google Scholar), the DNA intermediate in A is for the formation of Holliday known to exist during meiotic recombination (1Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar). we can only speculate as to the secondary structures the 3′ strand are not also upon by the complex. Genetic and biochemical evidence that the 3′ strand is or that the 5′ strand is We that the 3′ ssDNA as of the might be to by a of proteins that in function to the 3′ ssDNA the secondary structures that might be to the of such protein might be and other recombination that have been to to the of a DSB Y. M. Lee Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. EMBO 2000; 1: PubMed Scopus Google Scholar, Res. PubMed Scopus Google Scholar). We to that the is and be that Mre11 nuclease in DSB processing in We that the of the complex in in such is to a DNA to the a DNA is as a of DNA strand by the A, the that of Mre11 are capable of DSB processing L.S. S. Res. 2000; PubMed Scopus Google that the 5′ ssDNA strand in the is also to other as by the of Symington L.S. S. Res. 2000; PubMed Scopus Google Scholar). has been that in the of Mre11, DNA can be by a exonuclease L.S. S. Res. 2000; PubMed Scopus Google Scholar). the of 5′ strand in such is evidenced by L.S. S. Res. 2000; PubMed Scopus Google Scholar). implicates the as the for DSB processing in We for and for