Effect of Amino Acid Substitutions in the Rad50 ATP Binding Domain on DNA Double Strand Break Repair in Yeast
2004; Elsevier BV; Volume: 280; Issue: 4 Linguagem: Inglês
10.1074/jbc.m410192200
ISSN1083-351X
AutoresLing Chen, Kelly M. Trujillo, Stephen Van Komen, Dong Hyun Roh, Lumír Krejčí, L. Kevin Lewis, Michael A. Resnick, Patrick Sung, Alan E. Tomkinson,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoThe Saccharomyces cerevisiae Rad50-Mre11-Xrs2 complex plays a central role in the cellular response to DNA double strand breaks. Rad50 has a globular ATPase head domain with a long coiled-coil tail. DNA binding by Rad50 is ATP-dependent and the Rad50-Mre11-Xrs2 complex possesses DNA unwinding and endonuclease activities that are regulated by ATP. Here we have examined the role of the Rad50 Walker type A ATP binding motif in DNA double strand break repair by a combination of genetic and biochemical approaches. Replacement of the conserved lysine residue within the Walker A motif with alanine, glutamate, or arginine results in the same DNA damage sensitivity and homologous recombination defect as the rad50 deletion mutation. The Walker A mutations also cause a deficiency in non-homologous end-joining. As expected, complexes containing the rad50 Walker A mutant proteins are defective in ATPase, ATP-dependent DNA unwinding, and ATP-stimulated endonuclease activities. Although the DNA end-bridging activity of the Rad50-Mre11-Xrs2 complex is ATP-independent, the end-bridging activity of complexes containing the rad50 Walker A mutant proteins is salt-sensitive. These results provide a molecular explanation for the observed in vivo defects of the rad50 Walker mutant strains and reveal a novel ATP-independent function for Rad50 in DNA end-bridging. The Saccharomyces cerevisiae Rad50-Mre11-Xrs2 complex plays a central role in the cellular response to DNA double strand breaks. Rad50 has a globular ATPase head domain with a long coiled-coil tail. DNA binding by Rad50 is ATP-dependent and the Rad50-Mre11-Xrs2 complex possesses DNA unwinding and endonuclease activities that are regulated by ATP. Here we have examined the role of the Rad50 Walker type A ATP binding motif in DNA double strand break repair by a combination of genetic and biochemical approaches. Replacement of the conserved lysine residue within the Walker A motif with alanine, glutamate, or arginine results in the same DNA damage sensitivity and homologous recombination defect as the rad50 deletion mutation. The Walker A mutations also cause a deficiency in non-homologous end-joining. As expected, complexes containing the rad50 Walker A mutant proteins are defective in ATPase, ATP-dependent DNA unwinding, and ATP-stimulated endonuclease activities. Although the DNA end-bridging activity of the Rad50-Mre11-Xrs2 complex is ATP-independent, the end-bridging activity of complexes containing the rad50 Walker A mutant proteins is salt-sensitive. These results provide a molecular explanation for the observed in vivo defects of the rad50 Walker mutant strains and reveal a novel ATP-independent function for Rad50 in DNA end-bridging. DNA double strand breaks (DSBs) 1The abbreviations used are: DSB, DNA double-strand break; HR, homologous recombination; NHEJ, non-homologous end-joining; RMX, Rad50-Mre11-Xrs2; TLC, thin layer chromatography; AFM, atomic force microscopy; WT, wild type; HR, homologous recombination; Gy, Gray. arise from a variety of sources including normal physiological programs, such as mating type switching in yeast, and as a result of DNA damage. These lesions are highly cytotoxic and mutagenic, and their removal is mediated by homologous recombination (HR) and non-homologous end-joining (NHEJ) (1Krejci L. Chen L. Van Komen S. Sung P. Tomkinson A. Progress Nucleic Acid Res. Mol. Biol. 2003; 75: 159-201Crossref Scopus (58) Google Scholar). Genetic and biochemical studies have led to the identification of many components of these two DNA repair pathways. Although HR and NHEJ mostly involve unique sets of protein factors, the yeast Rad50-Mre11-Xrs2 (RMX) complex has been implicated in both pathways (1Krejci L. Chen L. Van Komen S. Sung P. Tomkinson A. Progress Nucleic Acid Res. Mol. Biol. 2003; 75: 159-201Crossref Scopus (58) Google Scholar). In addition, this evolutionarily conserved protein complex participates in telomere maintenance, DNA damage-activated cell cycle checkpoints, the release of Spo11 protein from meiotic DSBs, and possibly sister chromatid cohesion (2Boulton S.J. Jackson S.P. EMBO J. 1998; 17: 1819-1828Crossref PubMed Scopus (556) Google Scholar, 3D'Amours D. Jackson S.P. Genes Dev. 2001; 15: 2238-2249Crossref PubMed Scopus (183) Google Scholar, 4Grenon M. Gilbert C. Lowndes N.F. Nat. Cell Biol. 2001; 3: 844-847Crossref PubMed Scopus (155) Google Scholar, 5Moreau S. Ferguson J.R. Symington L. Mol. Cell. Biol. 1999; 19: 556-566Crossref PubMed Scopus (364) Google Scholar, 6Hopfner K.P. Craig L. Moncalian G. Zinkel R.A. 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Chem. 2003; 278: 48957-48964Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Genetic studies indicate that the Mre11 nuclease activity is important for the processing of meiotic DSBs made by Spo11 and possibly telomere maintenance (5Moreau S. Ferguson J.R. Symington L. Mol. Cell. Biol. 1999; 19: 556-566Crossref PubMed Scopus (364) Google Scholar, 11Lewis L.K. Storici F. Van Komen S. Calero S. Sung P. Resnick M.A. Genetics. 2004; 166: 1701-1703Crossref PubMed Scopus (69) Google Scholar). The nuclease function of the RMX complex is among several redundant activities that resect DSBs in mitotic cells to yield single-stranded DNA tails to be used by the HR machinery to initiate recombinational repair (1Krejci L. Chen L. Van Komen S. Sung P. Tomkinson A. Progress Nucleic Acid Res. Mol. Biol. 2003; 75: 159-201Crossref Scopus (58) Google Scholar). Accordingly, the DNA damage sensitivity of strains lacking a functional RMX complex can be suppressed by overexpressing ExoI, a 5′-to-3′ exonuclease (12Lewis L.K. Karthikeyan G. Westmorland J.W. Resnick M.A. Genetics. 2002; 160: 49-62Crossref PubMed Google Scholar). Since the Mre11 nuclease is not required for NHEJ (5Moreau S. Ferguson J.R. Symington L. Mol. Cell. Biol. 1999; 19: 556-566Crossref PubMed Scopus (364) Google Scholar), at least when DNA molecules have complementary ends, it appears that the RMX complex has another function in NHEJ. In congruence with this idea, the RMX complex possesses a robust DNA end-bridging activity likely to be important for intermolecular DNA end-joining by Dnl4/Lif1 (13Chen L. Trujillo K. Ramos W. Sung P. Tomkinson A.E. Mol. Cell. 2001; 8: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Rad50 is a member of the family of structural maintenance of chromosome (SMC) proteins. Biochemical and structural studies of Rad50 from Pyrococcus furiosus indicate that the N- and C-terminal segments of Rad50, which contain Walker A and Walker B ATP binding domains (14Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4269) Google Scholar), respectively, assemble into a functional catalytic domain (Rad50cd) (15Hopfner K.P. Karchner A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 798-800Abstract Full Text Full Text PDF Scopus (812) Google Scholar). The intervening heptad repeat regions form an intramolecular coiledcoil with Mre11 binding to the coiled-coil region adjacent to the Rad50cd (15Hopfner K.P. Karchner A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 798-800Abstract Full Text Full Text PDF Scopus (812) Google Scholar, 16de Jager M. van Noort J. van Gent D.C. Dekker C. Kanaar R. Wyman C. Mol. 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Cell. 1998; 93: 477-486Abstract Full Text Full Text PDF PubMed Scopus (1027) Google Scholar). In support of this notion, a physical interaction between Xrs2 and the Lif1 subunit of Dnl4/Lif1 has been demonstrated (13Chen L. Trujillo K. Ramos W. Sung P. Tomkinson A.E. Mol. Cell. 2001; 8: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Recent studies have revealed that Xrs2 is a DNA structure-specific-binding protein and that it is indispensable for DNA end engagement and ATP-dependent DNA unwinding by the RMX complex (10Trujillo K. Roh D.H. Chen L. Komen VanS. Tomkinson A. Sung P. J. Biol. Chem. 2003; 278: 48957-48964Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). We have employed a combination of genetic and biochemical approaches to determine how ATP influences the biological functions of Rad50 and the RMX complex. Importantly, mutations that alter the conserved lysine residue in the Walker type A motif of yeast Rad50 impair both HR and NHEJ. Biochemically, the rad50 Walker mutations inactivate the ATPase activity of the RMX complex and lead to the loss of the ATP-dependent DNA structure-specific endonuclease and DNA unwinding activities of this complex as well. Even though the DNA end binding and end-bridging activities of the RMX complex are not influenced by ATP, we demonstrate that the Walker A motif itself is important for DNA end-bridging and DNA end-joining when a physiological level of salt is present. Our results provide molecular information concerning the role of ATP in RMX functions and also implicate the region in Rad50 protein that encompasses the Walker A motif in DNA end-bridging and DNA end-joining independent of ATP. Construction of Yeast Strains—The strain W1588-4C (MATa ade2-1 can1-100 leu2-3,112 his3-11,15 ura3-1 trp1-1 RAD5) was used for the integration of mutant alleles at the RAD50 locus. The integrating plasmids with mutations in the codon for lysine 40 were constructed by site-directed mutagenesis of YIp5::RAD50 containing the RAD50 open reading frame inserted in the BamHI site and carrying the URA3 marker. The rad50 mutant alleles were sequenced to ensure that no unintended mutation had been introduced during handling. Plasmids harboring the rad50 mutations were linearized with BglII and used to transform W1588-4C. After selecting for Ura+ transformants, cells were grown non-selectively and then streaked on 5-FOA plates. FOA-resistant colonies were irradiated with γ-rays and sensitive clones were examined for the presence of the rad50 mutations by PCR amplification and sequencing of the genomic region surrounding the RAD50 K40 site. γ-Irradiation—This was supplied by a 137cesium source. Three cultures of each strain were grown overnight, diluted to a titer of 5 × 106 cells per ml, and shaken for 3 h at 30 °C prior to each assay to ensure that cells were in log phase. Plasmid/Chromosome Recombination Assays—To assess mitotic recombination proficiency, wild type and mutant derivatives of W1588-4C cells were transformed separately with 200 ng of control vector pRS313 (CEN/ARS HIS3) (28Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) or 400 ng of BclI-cut pLKL37Y (URA3 HIS3) integrating plasmid. The plasmid pLKL37Y was purified from the dam– Escherichia coli strain SCS110 (Stratagene) to allow cleavage by BclI, which is sensitive to dam methylation. BclI cleaves at nt 549 of HIS3 on pLKL37Y to produce a fragment that can recombine with the chromosomal his3-11,15 allele and generate URA3 HIS3 integrants (12Lewis L.K. Karthikeyan G. Westmorland J.W. Resnick M.A. Genetics. 2002; 160: 49-62Crossref PubMed Google Scholar). Recombination frequencies (His+ Ura+ recombinants per microgram of transforming DNA) were normalized to pRS313 (His+) transformation efficiencies using the same competent cell preparations. Three cultures of each strain were transformed and the results averaged. Recombination frequencies are shown as a percentage of the frequency of the isogenic wild-type strain. Plasmid End-joining Assay—These assays were performed essentially as described (29Teo S.H. Jackson S.P. EMBO J. 1997; 16: 4788-4795Crossref PubMed Scopus (232) Google Scholar). Strains W1588-4C; DR50 (rad50Δ); DHY201 (rad50 K40A); DHY202 (rad50 K40R), and DHY203 (rad50 K40E) were transformed separately with uncut pBTM116 (CEN, ADH, TRP1) and EcoRI-linearized pBTM116. The transformation efficiency for the linearized plasmid was normalized to the transformation efficiency obtained with the uncut supercoiled plasmid DNA, using 3–4 cultures for each strain. Protein Purification and Assembly of Wild Type and Mutant Variants of the RMX Complex—The Rad50, Mre11, and Xrs2 proteins were individually purified from yeast and assembled into the trimeric RMX complex as described previously (9Trujillo K. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 13Chen L. Trujillo K. Ramos W. Sung P. Tomkinson A.E. Mol. Cell. 2001; 8: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). For overexpression of the Rad50 K40A, K40E, and K40R proteins in yeast, the corresponding mutant alleles were placed under the control of the galactose-inducible GAL-PGK promoter in vector pPM231 (2μ, GAL-PGK, LEU-2d) (9Trujillo K. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). The resulting plasmids (pR50.K40A.1, pR50.K40E.1, and pR50.K40R.1) were each introduced into the protease-deficient yeast strain BJ5464 (MATα, ura3–52, trp-1, leu2Δ, his3Δ200, pep4::HIS3, prbΔ1.6R), and the mutant proteins purified by the same procedure devised for wild-type Rad50 (9Trujillo K. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Wild-type and mutant Rad50 proteins were assembled into protein complexes as described previously (9Trujillo K. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 13Chen L. Trujillo K. Ramos W. Sung P. Tomkinson A.E. Mol. Cell. 2001; 8: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). The concentration of proteins and protein complexes was determined by densitometric scanning of Coomassie Blue-stained 7.5% SDS-PAGE gels (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) that contained different loadings of the proteins and protein complexes with known amounts of bovine serum albumin as the comparison standard. ATPase Assay—Purified wild-type and mutant RMX complexes (1.2 μm of each) were incubated with 150 μm [γ-32P]ATP and ϕX174 (+) strand (22 μm nucleotides) at 30 °C in 10 μl of buffer A (30 mm Bis-Tris pH 7.0, 1 mm dithiothreitol, 50 mm KCl, and 100 μg/ml bovine serum albumin, and 5 mm MgCl2). At the indicated times, an aliquot (2 μl) was removed and mixed with an equal volume of 1% SDS to halt the reaction. The level of ATP hydrolysis was determined by thin layer chromatography (TLC), as described (31Petukhova G.S. Stratton S.A. Sung P. Nature. 1998; 393: 88-91Crossref PubMed Scopus (346) Google Scholar). DNA Substrates—To construct the substrate for the DNA binding experiments, the 5′-end of the 83-mer oligonucleotide: 5′-TTG ATA AGA GGT CAT TTT TGC GGA TGG CTT AGA GCT TAA TTG CTG AAT CTG GTG CTG TAG CTC AAC ATG TTT TAA ATA TGC AA-3′ was radiolabeled using T4 polynucleotide kinase (Promega) and [γ-32P]ATP (Amersham Biosciences). The polynucleotide kinase was inactivated by heating the reaction mixture to 75 °C for 15 min, and unincorporated nucleotide was removed with a Spin 30 column (Bio-Rad). The labeled oligonucleotide was annealed with its exact complement and the 83-base pair duplex was purified from a 10% native polyacrylamide gel by overnight diffusion at 4 °C into TAE buffer (40 mm Tris acetate, pH 7.5, 0.5 mm EDTA). To make topologically relaxed DNA, ϕX174 replicative form I DNA (Invitrogen, Life Technologies, Inc.) was treated with calf thymus topoisomerase I (Invitrogen), followed by purification of the relaxed DNA species from agarose gels containing ethidium bromide, as described (32Van Komen, S., Petukhova, G., Sigurdsson, S., Stratton, S., and Sung, P. (200) Mol. Cell 6, 563–572Google Scholar). The relaxed DNA was stored in TAE buffer. Topological DNA Unwinding Assay—Wild type and mutant RMX complexes (1 μm of each) were incubated with topologically relaxed DNA (3 μm nucleotides) for 5 min at 37 °C in 10 μl of Buffer A containing 2 mm ATP. Under these conditions, the nuclease activity of Mre11 is dormant (8Paull T.T. Gellert M. Mol. Cell. 1998; 1: 969-979Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 9Trujillo K. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Calf thymus topoisomerase I (3 units; Invitrogen) was then added, and incubation continued for an additional 10 min at 37 °C. The reactions were stopped and deproteinized by incubation with SDS (0.5%) and proteinase K (0.5 mg/ml) for 5 min at 37 °C. DNA species were resolved by electrophoresis in 0.9% agarose gels and stained with ethidium bromide. Endonuclease Assay—The DNA hairpin (HP2) with ssDNA overhangs was labeled at the 3′-end as described previously (9Trujillo K. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). This substrate (120 nm nucleotides) was incubated with 400 nm of the wild-type and mutant RMX complexes at 4 °C in 10 μl of Buffer A containing 1 mm ATP and 5 mm MnCl2 instead of MgCl2. After 40 min at 37 °C, the reaction was halted by the addition of SDS and proteinase K (0.2% and 0.5 mg/ml, respectively). After separation by denaturing polyacrylamide gel electrophoresis, radiolabeled DNA species were detected in the dried gel by phosphorimaging analysis (9Trujillo K. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Exonuclease Assay—A 3′-end-labeled 74-bp duplex DNA substrate was generated by the hybridization of complementary oligonucleotides as described previously (9Trujillo K. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). This duplex (7.4 μm nucleotides) was incubated with 20 nm of the wild type and mutant RMX complexes at 4 °C in 10 μl of Buffer A that contained 5 mm MnCl2 instead of MgCl2. At the times indicated, an aliquot (2 μl) was removed and deproteinized by incubation with SDS (0.2%) and proteinase K (0.5 mg/ml) for 10 min at room temperature. After separation by TLC, the plates were air-dried and 32P-labeled molecules detected by phosphorimaging analysis. In Vitro DNA End-joining Assay—Linear 400-bp DNA fragments with either 5′ or 3′ complementary single-stranded ends were generated and end-labeled as described (33Chen L. Trujillo K. Sung P. Tomkinson A.E. J. Biol. Chem. 2000; 275: 26196-26205Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Labeled DNA substrates (0.75 nm DNA molecules) were incubated with Dnl4/Lif1 (4 nm) and, where indicated, the wild-type RMX complex or one of the mutant RMX complexes (6 nm of each) in reaction mixtures (20 μl) containing 60 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 10 mm KCl, 1 mm ATP, 5 mm dithiothreitol, and 50 μg/ml bovine serum albumin at 25 °C. Reactions were deproteinized by phenol/chloroform extraction. After separation by agarose gel electrophoresis, the labeled DNA substrates and products were visualized by phosphorimaging analysis of the dried gel. Lambda Exonuclease Protection Assay—A 400-bp duplex with complementary 5′ single-stranded ends was labeled with 32P as described (33Chen L. Trujillo K. Sung P. Tomkinson A.E. J. Biol. Chem. 2000; 275: 26196-26205Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The labeled substrate (38 nm DNA ends) was incubated with or without 100 nm of the wild-type and mutant RMX complexes in 10 μlof 30 mm Tris-HCl (pH 7.4), 1 mm dithiothreitol, 5 mm MgCl2, 0.3 mg/ml bovine serum albumin, and 50 mm KCl. After 5 min at room temperature, λ exonuclease (0.1 unit) was added to the reaction mixture. At the indicated times, an aliquot (1.5 μl) was removed and treated with SDS (0.2%) and proteinase K (0.5 mg/ml) to deproteinize the reaction. After separation by TLC, the 32P-labeled molecules were detected by phosphorimaging analysis. DNA Mobility Shift Assays—The indicated amounts of the wild-type and mutant RMX complexes were incubated for 10 min at 37 °C with the radiolabeled 83-mer duplex substrate (1.5 μm nucleotides) in 10 μl buffer A containing 2 mm ATP. The reaction mixtures were analyzed in 9% native polyacrylamide gels run in Tris acetate/EDTA buffer at 4 °C. The gels were dried and subjected to phosphorimaging analysis. Where indicated, deproteinization was done by incubating reaction mixtures with SDS (0.2%) and proteinase K (0.5 mg/ml) at 37 °C for 5 min. Atomic Force Microscopy (AFM)—AFM was performed using a Nanoscope Scanning Probe system (Digital Instruments Inc.) in the tapping mode, as described previously (33Chen L. Trujillo K. Sung P. Tomkinson A.E. J. Biol. Chem. 2000; 275: 26196-26205Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). A 400-bp DNA fragment with 5′-cohesive ends (20 nm) and either wild-type or mutant RMX complex (25 nm of each) were incubated in 15 μl of 50 mm Tris-HCl (pH 7.2), 1 mm dithiothreitol, 10 mm MgCl2, and the indicated concentration of KCl on ice for 10 min. Subsequently, the sample was diluted with an equal volume of 20 mm HEPES (pH 7.9) and 10 mm MgCl2 and applied to freshly cleaved mica. After 1 min at room temperature, the mica was washed with filter-sterilized distilled water and dried with a stream of filtered air before being imaged. Mutations in the Walker A Motif of Rad50 Cause Hypersensitivity to DNA-damaging Agents—Rad50 protein is a DNA-dependent ATPase (9Trujillo K. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) and binds DNA in an ATP-dependent manner (18Raymond W.E. Kleckner N. Nucleic Acids Res. 1993; 21: 3851-3856Crossref PubMed Scopus (100) Google Scholar). To elucidate how ATP regulates the functions of Rad50 and the RMX complex, we changed the conserved lysine residue (Lys40) in the Walker A motif of Rad50 to arginine (K40R), alanine (K40A), and glutamic acid (K40E) by site-directed mutagenesis. Yeast strains in which the RAD50 gene was replaced by the three mutant alleles were as sensitive as the isogenic rad50Δ strain to killing by ionizing radiation (IR) (Fig. 1A) and by methyl methanesulfonate (Fig. 1C). As expected, the DNA damage sensitivity of the rad50 mutants was fully complemented by the wild-type RAD50 gene but not the XRS2 gene (Fig. 1B). By contrast, replacement of the RAD50 gene with the rad50S allele (K81I), which is defective in the removal of Spo11 from DNA double strand breaks during meiosis (34Cao L. Alani E. Kleckner N. Cell. 1990; 61: 1089-1101Abstract Full Text PDF PubMed Scopus (537) Google Scholar), did not confer increased sensitivity to DNA damage (data not shown). Recently, it was shown that overexpression of ExoI, a 5′-to-3′ exonuclease (35Tishkoff D.X. Boerger A.L. Bertrand P. Filosi N. Gaida G.M. Kane M.F. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7487-7492Crossref PubMed Scopus (334) Google Scholar), suppressed the DNA damage hypersensitivity of rad50, mre11, and xrs2 strains (12Lewis L.K. Karthikeyan G. Westmorland J.W. Resnick M.A. Genetics. 2002; 160: 49-62Crossref PubMed Google Scholar). Overexpression of ExoI also suppressed the DNA damage hypersensitivity of rad50 K40A, rad50 K40R, and rad50 K40E strains although not quite to the same extent as the rad50Δ strain (Fig. 1C), suggesting that the presence of a defective Rad50-Mre11-Xrs2 complex may hinder ExoI activity. rad50 Walker A Mutants Are Compromised for Recombinational DNA Repair—Since inactivation of NHEJ in Saccharomyces cerevisiae does not cause an appreciable increase in sensitivity to ionizing radiation, it appears that defects in recombinational repair mechanisms underlie the hypersensitivity of rad50 strains (1Krejci L. Chen L. Van Komen S. Sung P. Tomkinson A. Progress Nucleic Acid Res. Mol. Biol. 2003; 75: 159-201Crossref Scopus (58) Google Scholar, 26Critchlow S.E. Jackson S.P. Trends Biochem. Sci. 1998; 23: 394-398Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 36Lewis L.K. Resnick M.A. Mutat. Res. 2000; 451: 71-89Crossref PubMed Scopus (145) Google Scholar). Based on the survival curves of the rad50 Walker mutant strains, we predicted that these strains would also be defective in homologous recombination. To test this idea, we used an assay that measures homologous integration of a linearized plasmid molecule (12Lewis L.K. Karthikeyan G. Westmorland J.W. Resnick M.A. Genetics. 2002; 160: 49-62Crossref PubMed Google Scholar). As anticipated, the rad50 K40A, rad50 K40R, and rad50 K40E mutants exhibited a marked defect in plasmid integration similar to that of the rad50Δ strain (Fig. 2A). In contrast, the rad50S strain was only slightly impaired (data not shown). Rad50 Walker A Mutants Are Defective in DNA End-joining—Although inactivation of NHEJ in yeast does not cause a significant increase in sensitivity to DNA damaging agents, defects in this pathway do result in a reduced ability to recircularize linearized plasmid DNA molecules (2Boulton S.J. Jackson S.P. EMBO J. 1998; 17: 1819-1828Crossref PubMed Scopus (556) Google Scholar, 26Critchlow S.E. Jackson S.P. Trends Biochem. Sci. 1998; 23: 394-398Abstract Full Text Full Text PDF P
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