hXRCC2 Enhances ADP/ATP Processing and Strand Exchange by hRAD51
2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês
10.1074/jbc.m306066200
ISSN1083-351X
AutoresKang Sup Shim, Christoph Schmütte, Gregory Tombline, Christopher D. Heinen, Richard Fishel,
Tópico(s)CRISPR and Genetic Engineering
ResumoThe assembly of bacterial RecA, and its human homolog hRAD51, into an operational ADP/ATP-regulated DNA-protein (nucleoprotein) filament is essential for homologous recombination repair (HRR). Yet hRAD51 lacks the coordinated ADP/ATP processing exhibited by RecA and is less efficient in HRR reactions in vitro. In this study, we demonstrate that hXRCC2, one of five other poorly understood non-redundant human mitotic RecA homologs (hRAD51B, hRAD51C, hRAD51D, hXRCC2, and hXRCC3), stimulates hRAD51 ATP processing. hXRCC2 also increases hRAD51-mediated DNA unwinding and strand exchange activities that are integral for HRR. Although there does not seem to be a long-lived interaction between hXRCC2 and hRAD51, we detail a strong adenosine nucleotide-regulated interaction between the hXRCC2-hRAD51D heterodimer and hRAD51. These observations begin to elucidate the separate and specialized functions of the human mitotic RecA homologs that enable an efficient nucleoprotein filament required for HRR. The assembly of bacterial RecA, and its human homolog hRAD51, into an operational ADP/ATP-regulated DNA-protein (nucleoprotein) filament is essential for homologous recombination repair (HRR). Yet hRAD51 lacks the coordinated ADP/ATP processing exhibited by RecA and is less efficient in HRR reactions in vitro. In this study, we demonstrate that hXRCC2, one of five other poorly understood non-redundant human mitotic RecA homologs (hRAD51B, hRAD51C, hRAD51D, hXRCC2, and hXRCC3), stimulates hRAD51 ATP processing. hXRCC2 also increases hRAD51-mediated DNA unwinding and strand exchange activities that are integral for HRR. Although there does not seem to be a long-lived interaction between hXRCC2 and hRAD51, we detail a strong adenosine nucleotide-regulated interaction between the hXRCC2-hRAD51D heterodimer and hRAD51. These observations begin to elucidate the separate and specialized functions of the human mitotic RecA homologs that enable an efficient nucleoprotein filament required for HRR. RecA is the prototypical mediator of homologous recombination repair (HRR) 1The abbreviations used are: HRR, homologous recombination repair; NTP, nucleotide triphosphate; NPF, nucleoprotein filament; GEF, guanine nucleotide exchange factors; XRCC, x-ray sensitive cross complementation group; nt, nucleotide; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; RFI, replicative form I; GST, glutathione S-transferase; IVTT, in vitro transcription/translation; ATPγS, adenosine-5′-O-(3-thio)triphosphate; ATPase, ATP hydrolysis activity; jm, joint molecule; ϕX174, bacteriophage X174. in bacteria (for review, see Refs. 1Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar and 2Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar). The complexity of HRR is dramatically increased in human cells, where six non-redundant mitotic RecA homologs (hRAD51, hRAD51B, hRAD51C, hRAD51D, hXRCC2, and hXRCC3) are required for efficient HRR in vivo (3Sonoda E. Sasaki M.S. Morrison C. Yamaguchi Iwai Y. Takata M. Takeda S. Mol. Cell. Biol. 1999; 19: 5166-5169Crossref PubMed Scopus (373) Google Scholar, 4Takata M. Sasaki M.S. Tachiiri S. Fukushima T. Sonoda E. Schild D. Thompson L.H. Takeda S. Mol. Cell. Biol. 2001; 21: 2858-2866Crossref PubMed Scopus (467) Google Scholar). Unlike the other human RecA/RAD51 family members, RAD51 seems to be essential for HRR in both mitotic and meiotic cells (5Shinohara A. Ogawa T. Mutat. Res. 1999; 435: 13-21Crossref PubMed Scopus (64) Google Scholar, 6Sung P. Trujillo K.M. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (161) Google Scholar), suggesting a fundamental role in eukaryotic HRR. The RecA/RAD51 family of proteins promotes HRR by catalyzing homologous pairing and strand exchange between parental DNAs (2Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar, 5Shinohara A. Ogawa T. Mutat. Res. 1999; 435: 13-21Crossref PubMed Scopus (64) Google Scholar, 6Sung P. Trujillo K.M. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (161) Google Scholar, 7Kowalczykowski S.C. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 539-575Crossref PubMed Scopus (243) Google Scholar). A number of discrete heteromeric complexes between the human proteins have been identified (8Schild D. Lio Y. Collins D.W. Tsomondo T. Chen D.J. J. Biol. Chem. 2000; 275: 16443-16449Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Whereas hRAD51 and a hRAD51C-hXRCC3 complex seem to retain a homologous pairing and strand exchange activity (9Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 10Gupta R.C. Bazemore L.R. Golub E.I. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 463-468Crossref PubMed Scopus (241) Google Scholar, 11Kurumizaka H. Ikawa S. Nakada M. Eda K. Kagawa W. Takata M. Takeda S. Yokoyama S. Shibata T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5538-5543Crossref PubMed Scopus (114) Google Scholar, 12New J.H. Sugiyama T. Zaitseva E. Kowalczykowski S.C. Nature. 1998; 391: 407-410Crossref PubMed Scopus (510) Google Scholar, 13Masson J.Y. Stasiak A.Z. Stasiak A. Benson F.E. West S.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8440-8446Crossref PubMed Scopus (111) Google Scholar, 14Mazin A.V. Bornarth C.J. Solinger J.A. Heyer W.D. Kowalczykowski S.C. Mol. Cell. 2000; 6: 583-592Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the function(s) of the remaining human RecA/RAD51 homologs is unknown. Homology between RecA/RAD51 family members is largely confined to the Walker A/B nucleotide binding domains (2Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar, 7Kowalczykowski S.C. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 539-575Crossref PubMed Scopus (243) Google Scholar, 15Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4388) Google Scholar). These peptide motifs allow a number of diverse proteins to coordinate the free energy of nucleotide triphosphate (NTP) binding and hydrolysis into biological processes (15Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4388) Google Scholar). During HRR, the bacterial RecA protein efficiently synchronizes the binding and hydrolysis of ATP between monomers within a nucleoprotein filament (NPF) that ultimately facilitates unwinding and strand exchange between homologous DNAs (2Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar, 7Kowalczykowski S.C. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 539-575Crossref PubMed Scopus (243) Google Scholar). However, hRAD51 alone is largely unable to coordinate ATP processing between individual subunits of the NPF, which is manifest in modest strand exchange activity and a dramatically reduced ability to process heterologous (mismatched) DNA sequences, a genetic signature of HRR (9Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 10Gupta R.C. Bazemore L.R. Golub E.I. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 463-468Crossref PubMed Scopus (241) Google Scholar, 16Sung P. Robberson D.L. Cell. 1995; 82: 453-461Abstract Full Text PDF PubMed Scopus (434) Google Scholar, 17Gupta R.C. Folta-Stogniew E. O'Malley S. Takahashi M. Radding C.M. Molecular Cell. 1999; 4: 705-714Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 18Namsaraev E.A. Berg P. J. Biol. Chem. 2000; 275: 3970-3976Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 19Sigurdsson S. Trujillo K. Song B. Stratton S. Sung P. J. Biol. Chem. 2001; 276: 8798-8806Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20Tombline G. Fishel R. J. Biol. Chem. 2002; 277: 14417-14425Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Tombline G. Shim K.S. Fishel R. J. Biol. Chem. 2002; 277: 14426-14433Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 22Tombline G. Heinen C.D. Shim K.S. Fishel R. J. Biol. Chem. 2002; 277: 14434-14442Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 23Rice K.P. Eggler A.L. Sung P. Cox M.M. J. Biol. Chem. 2001; 276: 38570-38581Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 24Holmes V.F. Benjamin K.R. Crisona N.J. Cozzarelli N.R. Nucleic Acids Res. 2001; 29: 5052-5057Crossref PubMed Scopus (24) Google Scholar). These results have suggested that additional factors are necessary to coordinate the hRAD51 ATPase during HRR in eukaryotic cells. Hydrolysis of an NTP can be conceptually divided into two phases: 1) γ-phosphate hydrolysis and 2) the release of the hydrolysis products (NDP + Pi) followed by binding of a new NTP (NDP→NTP exchange). NDP→NTP exchange seems to be the rate-limiting step in many NTPase cycles (25Vale R.D. J. Cell Biol. 1996; 135: 291-302Crossref PubMed Scopus (247) Google Scholar, 26Fishel R. Genes Dev. 1998; 12: 2096-2101Crossref PubMed Scopus (154) Google Scholar, 27Sablin E.P. Fletterick R.J. Curr. Opin. Struct. Biol. 2001; 11: 617-724Crossref PubMed Scopus (50) Google Scholar). The regulation of NDP→NTP exchange by “exchange factors” is one mechanism by which cells may control protein conformational transitions that are coupled to biological function. Such regulated control seems to enhance the efficiency of NTPases (28Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (896) Google Scholar). Examples of exchange factors and their cognate NTPases include guanine nucleotide exchange factors (GEFs) for G-proteins, profilin for actin, actin for myosin, and β-tubulin for dynein and kinesin (25Vale R.D. J. Cell Biol. 1996; 135: 291-302Crossref PubMed Scopus (247) Google Scholar, 26Fishel R. Genes Dev. 1998; 12: 2096-2101Crossref PubMed Scopus (154) Google Scholar, 27Sablin E.P. Fletterick R.J. Curr. Opin. Struct. Biol. 2001; 11: 617-724Crossref PubMed Scopus (50) Google Scholar). Biochemical evidence that links exchange factors with ATPases has been previously inferred from single-turnover ATP hydrolysis studies and/or comparison of protein-cofactor alterations of ADP/ATP binding (for example, see Refs. 29Kabani M. Beckerich J.M. Brodsky J.L. Mol. Cell. Biol. 2002; 22: 4677-4689Crossref PubMed Scopus (119) Google Scholar and 30Kabani M. Beckerich J.M. Gaillardin C. Mol. Cell. Biol. 2000; 20: 6923-6934Crossref PubMed Scopus (72) Google Scholar). It is noteworthy that there are very few examples in which a direct examination of ADP→ATP exchange has been demonstrated (31Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 32Easter Jr., J. Gober J.W. Mol. Cell. 2002; 10: 427-434Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The human mitotic RecA homolog XRCC2 (x-ray sensitive cross complementation group-2) was identified based on its ability to complement the sensitivity of irs1 hamster cells to the DNA cross-linking agent mitomycin C (33Jones N.J. Cox R. Thacker J. Mutat. Res. 1987; 183: 279-286Crossref PubMed Scopus (186) Google Scholar, 34Thacker J. Trends Genet. 1999; 15: 166-168Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Although the biochemical and molecular basis of XRCC2 function is unknown, these studies have suggested that it plays an important role in HRR (35Liu N. Lamerdin J.E. Tebbs R.S. Schild D. Tucker J.D. Shen M.R. Brookman K.W. Siciliano M.J. Walter C.A. Fan W. Narayana L.S. Zhou Z.Q. Adamson A.W. Sorensen K.J. Chen D.J. Jones N.J. Thompson L.H. Mol. Cell. 1998; 1: 783-793Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 36Johnson R.D. Liu N. Jasin M. Nature. 1999; 401: 397-399Crossref PubMed Google Scholar). In this study, we demonstrate that hXRCC2 enhances ATP processing by hRAD51. Unlike other well-characterized RecA/RAD51 homologs, hXRCC2 does not seem to possess significant intrinsic DNA binding, ADP/ATP binding, or ATPase activities. We confirm and purify a stable hXRCC2-hRAD51D heterodimer (37Masson J.Y. Tarsounas M.C. Stasiak A.Z. Stasiak A. Shah R. McIlwraith M.J. Benson F.E. West S.C. Genes Dev. 2001; 15: 3296-3307Crossref PubMed Scopus (309) Google Scholar, 38Kurumizaka H. Ikawa S. Nakada M. Enomoto R. Kagawa W. Kinebuchi T. Yamazoe M. Yokoyama S. Shibata T. J. Biol. Chem. 2002; 277: 14315-14320Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). hRAD51D also does not seem to significantly bind ATP. We find that hRAD51D and the hXRCC2-hRAD51D heterodimer only interact with hRAD51 in the presence of the reaction intermediate mimetic ADP-aluminum fluoride. These results are consistent with a role for hRAD51D in localizing hXRCC2 to the active site of the hRAD51 NPF and a unique role for hXRCC2 in regulating hRAD51 activities. Protein Purification—hRAD51 and hRAD51(K133R) [K/R hRAD51] was purified as described previously (20Tombline G. Fishel R. J. Biol. Chem. 2002; 277: 14417-14425Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The cDNA encoding the C-terminal His6-tagged hXRCC2 was subcloned into pFast-Bac-Dual (BAC-TO-BAC baculovirus expression system; Invitrogen). The hRAD51D was inserted into the second expression site of pFast-Bac-Dual vector containing His6-hXRCC2. hXRCC2 and the hXRCC2-hRAD51D heterodimer were overexpressed in High Five insect cells (Invitrogen). Infected cells containing overexpressed hXRCC2 or hXRCC2-hRAD51D were harvested and resuspended in buffer A (20 mm HEPES-NaOH, pH 7.5, 300 mm NaCl, 10% glycerol, 20 mm imidazole, and protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 0.8 μg/ml leupeptin, 0.8 μg/ml pepstatin)) followed by rapid freezing in liquid nitrogen. Cell lysates were prepared by thawing cells on ice, passed them through a 25-gauge needle, and clearing debris by ultracentrifugation. For hXRCC2 purification the supernatant was loaded onto a nickel-nitrilotriacetic acid Superflow (Qiagen) column, washed with buffer A, and eluted with a linear gradient of imidazole from 20 mm to 200 mm. Pooled fractions (fraction I) containing hXRCC2 were dialyzed against buffer B (20 mm HEPES-NaOH, pH 7.5, 150 mm NaCl, 10% glycerol, 1 mm dithiothreitol, and protease inhibitors). Fraction I was loaded on to Mono-S in tandem with a Heparin-Sepharose column (Amersham Biosciences). The flow through (fraction II) was dialyzed against buffer C (5 mm potassium phosphate, pH 6.8, 150 mm NaCl, 10% glycerol, 1 mm dithiothreitol, and protease inhibitors). Fraction II was loaded onto a hydroxylapatite column in tandem with Polybuffer Exchanger (PBE) (Pharmacia). The PBE column was disconnected. The remaining hydroxyapatite column, including hXRCC2 protein, was washed and eluted with a linear gradient of potassium phosphate from 5 to 200 mm. Pooled fractions (fraction III) were dialyzed against 20 mm HEPES-NaOH, pH 7.5, 150 mm NaCl, 10% glycerol, 1 mm dithiothreitol, 0.1 mm EDTA, and protease inhibitors, snap-frozen, and stored at –80 °C. Fraction III hXRCC2 was stable for several months. To purify the hXRCC2-hRAD51D heterodimer, the cleared cellular supernatant was loaded and eluted from a nickel-nitrilotriacetic acid Superflow (Qiagen) column as above. Pooled fractions (fraction I) containing hXRCC2-hRAD51D were dialyzed against buffer B and loaded onto a tandem MonoS (Pharmacia)/Heparin-Agarose (Amersham Biosciences) column. The flow through (fraction II) was collected and loaded directly onto Hydroxyapatite (ceramic Micro-Prep; Bio-Rad) and eluted in buffer B with a 0–400 mm potassium phosphate gradient. Pooled fractions (fraction III) eluting at ∼200 mm potassium phosphate were collected and dialyzed against buffer B, loaded onto a Mono Q Column (Pharmacia), and eluted with a linear sodium chloride gradient (150–1000 mm). Pooled fractions (fraction IV) eluting at ∼250 mm NaCl were dialyzed against buffer B, frozen and stored at –80 °C. ATPase and ATPγS Binding—The ATPase activity was measured in 10 μl buffer X (20 mm HEPES-NaOH, pH 7.5, 150 mm NaCl, 10% glycerol, 2 mm MgCl2,1mm dithiothreitol, 0.1 mm EDTA, and 100 μg/ml bovine serum albumin), plus 3 μm (nt or bp) ϕX174 ssDNA or ϕX174 dsDNA replicative form I (RFI) (unless otherwise specified) and the indicated concentration of ATP/[γ-32P]ATP. Reactions were incubated at 37 °C for 30 min and stopped by the addition of 400 μl of 10% activated charcoal (Sigma) containing 1 mm EDTA (20Tombline G. Fishel R. J. Biol. Chem. 2002; 277: 14417-14425Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The charcoal was pelleted and duplicate 50 μl aliquots of the supernatant were counted by the Cherenkov method. ATPγS binding was performed by incubating 1 μm hXRCC2 with the indicated amount of ATPγS for 30 min in buffer X supplemented with 2 mm Mg(OAc)2 and subsequently placed on ice for 20 min. 4 ml of ice-cold buffer X supplemented with 2 mm Mg(OAc)2 was added, and samples were filtered (HAWP filters; Millipore). The filters were dried and counted by liquid scintillation as described previously (31Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Gel Mobility Shift—The single-stranded oligonucleotide dT50 (oligo-dT50)was synthesized and 5′ end-labeled with [γ-32P]ATP. Labeled substrates were gel-purified and DNA concentrations are expressed in moles of nucleotide. 41 bp of oligonucleotide dsDNA was prepared as described previously (31Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Gel mobility shift assay was performed in 20 μl of buffer X supplemented with 2 mm Mg(OAc)2 and contained labeled oligo-dT50 ssDNA or 41 bp of dsDNA, hRAD51, and indicated concentration of hXRCC2. Concentrations of adenosine nucleotides are indicated in the figure legends. The reactions were incubated at 37 °C for 30 min unless otherwise indicated. Protein-DNA complexes were resolved by 4% nondenaturing PAGE in 1× Tris-borate/EDTA buffer. Gels were dried and exposed to PhosphorImager screens (Amersham Biosciences) to visualize the products as described previously (31Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). ADP Binding and ADP→ATP Exchange—ADP binding activity was measured in 10 μl of buffer X supplemented with 2 mm Mg(OAc)2,3 μm (nt or bp) ϕX174 ssDNA or ϕX174 dsDNA RFI (unless otherwise specified), the indicated concentration of ADP in Fig. 3 containing 2 μm [3H]ADP. Reactions were incubated at 37 °C for 1 h and placed on ice for 10 min. The solution was filtered through a nitrocellulose membrane (HAWP; Millipore) and washed with 4 ml of ice-cold buffer X supplemented with 2 mm Mg(OAc)2. Filters were air-dried, incubated overnight in scintillation fluid, and the amount of radioactivity retained on the filters was determined as described previously (31Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). The ADP→ATP exchange was measured in buffer X supplemented with 2 mm Mg(OAc)2 plus indicated ADP/[3H]ADP. 0.6 μm hRAD51 was pre-incubated with ADP and ϕX174 DNA at 37 °C for 15 min in a final volume of 10 μl. ADP→ATP exchange was initiated by adding the indicated concentration of hXRCC2, and 1 mm ATP in buffer X supplemented with 2 mm Mg(OAc)2 (final volume, 30 μl), and incubation was continued at 25 °C. Reactions were stopped at indicated times in Fig. 3 by dilution and immediate filtration through a nitrocellulose membrane (HAWP; Millipore). Radioactivity retained on the filters was quantitated as described previously (31Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). UV Cross-linking—Reactions were performed as described previously (21Tombline G. Shim K.S. Fishel R. J. Biol. Chem. 2002; 277: 14426-14433Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In brief, 1 μm hXRCC2 and 1 μm hRAD51 were incubated at 25 °C for 15 min. in 10 μl of buffer X supplemented with 1 μm [α-32P]ATP (60 Ci/mmol) in the presence of 6 μm ϕX174 ssDNA and 2 mm Mg(OAc)2. The plate was irradiated at 254 nm in a Stratalinker (Stratagene) for 10 min. Samples were resolved by 10% SDS-PAGE and proteins were visualized by Coomassie stain. After digital imaging (Epson Perfection 636), the gel was dried and radiolabel visualized with a PhosphorImager. DNA Unwinding—DNA unwinding catalyzed by hRAD51 was examined using a modification of a method described previously (14Mazin A.V. Bornarth C.J. Solinger J.A. Heyer W.D. Kowalczykowski S.C. Mol. Cell. 2000; 6: 583-592Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Relaxed DNA (form IV) was prepared in batch by incubating 9 μg of ϕX174 replicative form I DNA (New England Biolabs) with 30 units of calf thymus topoisomerase I (Invitrogen) in 40 μl at 37 °C for 40 min. DNA unwinding assays were then initiated by addition of 2 μl (34 μm nucleotides) of batch prepared relaxed ϕX174 DNA, including topoisomerase I, to the indicated amount of hRAD51 and/or hXRCC2 in 20 μl of buffer X supplemented with 10 mm Mg(OAc)2 and 5 mm ATP or ADP at 37 °C for 10 min. The reactions were deproteinized by adding 2 μl of 10% SDS and 15 mg/ml proteinase K and incubated at 37 °C for 20 min. 2 μl of loading buffer (0.25% bromphenol blue/0.25% xylene cyanol and 50% glycerol) was added and 24 μl of the final sample volume subjected to electrophoresis in 1% agarose gel in Tris-acetate/EDTA buffer and followed by ethidium bromide (0.5 μg/ml) staining. Strand Exchange—Reactions were performed as described previously (19Sigurdsson S. Trujillo K. Song B. Stratton S. Sung P. J. Biol. Chem. 2001; 276: 8798-8806Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) with some modification. Linear ϕX174 dsDNA were prepared by digestion of ϕX174 RFI dsDNA with ApaL1. All the reaction steps were carried out at 37 °C. The reaction was assembled by mixing 6.0 μm hRAD51 (16 μl) and 30 μm (nt) circular ϕX174 ssDNA ± 1.0 μm hXRCC2 (2.6 μl) in 80 μl of final reaction volume (20 mm HEPES-NaOH, pH 7.5, 1 mm Mg(OAc)2,2mm ATP, and 1 mm dithiothreitol) for Fig. 5B (otherwise in the presence of indicated amount of hXRCC2 for Fig. 5C). After 5 min of incubation, (NH4)2SO4 (final concentration, 100 mm) and linear duplex DNA (final concentration, 15 μm) were added sequentially. At the indicated times in Fig. 3, 10-μl aliquots were withdrawn, the reaction was stopped and deproteinized by adding 3 μl of 10% SDS and 15 mg/ml proteinase K, incubated further for 20 min, and subjected to electrophoresis in 0.9% agarose gels containing 0.5 μg/ml ethidium bromide in Tris-acetate/EDTA buffer. GST-IVTT Interaction—Reactions were performed as described previously (39Guerrette S. Wilson T. Gradia S. Fishel R. Mol. Cell. Biol. 1998; 18: 6616-6623Crossref PubMed Scopus (122) Google Scholar, 40Schmutte C. Sadoff M.M. Shim K.S. Acharya S. Fishel R. J. Biol. Chem. 2001; 276: 33011-33018Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Full-length hRAD51 cDNA was subcloned into pGEX4T-2 (Pharmacia), which allows high expression of a glutathione S-transferase (GST) fusion protein. The fusion product was expressed in Escherichia coli, and an extract was generated as described previously (40Schmutte C. Sadoff M.M. Shim K.S. Acharya S. Fishel R. J. Biol. Chem. 2001; 276: 33011-33018Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The cleared extract was incubated with glutathione-agarose beads (Sigma) at 4 °C for 1 h under gentle continuous agitation and subsequently centrifuged. Each pellet was washed three times with 500 μl of binding buffer (20 mm Tris, pH 7.5, 10% glycerol, 150 mm NaCl, 5 mm EDTA or 10 mm MgCl2, 1 mm DTT, 0.1% Tween 10, 0.75 mg/ml bovine serum albumin, 0.5 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin). Under these conditions, ∼20–50 ng of protein was bound to 25 μl of beads. Lysates containing the unmodified pGEX vector were treated in the same way and used as a negative control. GST and GST-fusion protein binding to the beads was verified by denaturing gel electrophoresis (SDS-PAGE). 35S-labeled hRAD51D was synthesized using in vitro transcription/translation (IVTT) (TnT coupled reticulocyte lysate system; Promega) and added to the GST fusion protein-bound beads in binding buffer. ADP, ATPγS, ATP, and NaAlF4 (1 mm) were added as indicated. The samples were gently rocked at 4 °C for 1 h. The beads were then centrifuged, washed three times, as above, and the bound proteins were separated by PAGE and detected using a PhosphorImager system. No binding of the IVTT material to unmodified GST was detected under any of the conditions described previously (data not shown). Immunoprecipitation—Protein A beads (Sigma) suspended in buffer I (25 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm EDTA, and 20 mm dithiothreitol) were exposed over night to hRAD51 polyclonal antibody. The Protein A beads were then washed with binding buffer (20 mm Tris, pH 7.5, 10% glycerol, 150 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 0.1% Tween 10, 0.75 mg/ml bovine serum albumin, 0.5 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin) and incubated at 4 °C for 1h with purified hRAD51 with hXRCC2 or the heterodimer hRAD51D/XRCC2 as indicated. ADP and NaAlF4 (1 mm) were added as indicated. After three washes with binding buffer, the bound proteins were subjected to PAGE and detected by Western blot using monoclonal antibodies to hRAD51 and XRCC2 (Novus). IAsys Biosensor DNA-protein Interaction—IAsys Biosensor studies were performed using an IAsys Auto+ unit (Affinity Sensors, Cambridge, UK). A model oligonucleotide (oligo-dT50) with 5′-end biotinylated (Glen Research, Sterling, VA) was attached via streptavidin to the surface of an IAsys SPR cuvette pre-coated with biotin (Affinity Sensors). The kinetics of wild-type hRAD51 (250 nm) and K/R hRAD51 (250 nm) DNA binding ± hXRCC2 (250 nm) were measured in 20 mm HEPES-NaOH, pH 7.5, 150 mm NaCl, 5% glycerol, 2 mm MgCl2, and 1 mm dithiothreitol. Where indicated, 2.5 mm ADP, ATP, or ATPγS were added to the binding mixture. Representative binding isotherms are shown. Purification and Characterization of hXRCC2—To define its biochemical function, we have purified human XRCC2 (hXRCC2) to apparent homogeneity (Fig. 1A). Unlike bacterial RecA or eukaryotic RAD51 proteins (2Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar, 9Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 10Gupta R.C. Bazemore L.R. Golub E.I. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 463-468Crossref PubMed Scopus (241) Google Scholar, 20Tombline G. Fishel R. J. Biol. Chem. 2002; 277: 14417-14425Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 41Benson F.E. Stasiak A. West S.C. EMBO J. 1994; 13: 5764-5771Crossref PubMed Scopus (403) Google Scholar), hXRCC2 did not seem to bind ssDNA or dsDNA (Fig. 1B). DNA binding was not observed with different length DNA substrates (oligo-dT50 or ϕX174 and up to 30 μm ssDNA; data not shown), in the absence/presence of adenosine nucleotide (AMP, ADP, ATP, ATPγS; data not shown), or at hXRCC2 concentrations that were nearly 10-fold in excess of those required for complete binding by hRAD51 (Figs. 1B and 2A, lane 6; data not shown). We also evaluated the ability of hXRCC2 to bind and hydrolyze adenosine nucleotides. hXRCC2 displayed an extremely weak steady-state ATP hydrolysis (ATPase) activity (Fig. 1C; Table I) and ATPγS binding activity that seemed to be largely independent of DNA (Fig. 1D; Table I). hXRCC2 displayed negligible ATP binding and no effect on hRAD51 ATP binding in the presence of up to 1 mm nucleotide. The catalytic efficiency (kcat/Km) of the hXRCC2 ATPase was ∼100-fold less than hRAD51 and 2500-fold less than RecA (Table I; Ref. 20Tombline G. Fishel R. J. Biol. Chem. 2002; 277: 14417-14425Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To confirm the ATPγS binding results we performed ATP-cross-linking studies (Fig. 1E). In agreement with previous studies, we observed strong cross-linking of ATP to hRAD51 (21Tombline G. Shim K.S. Fishel R. J. Biol. Chem. 2002; 277: 14426-14433Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and negligible cross-linking of ATP to hXRCC2 (Fig. 1E). We were unable to detect significant ADP binding by hXRCC2 (Fig. 1F, ×; Table I). These results highlight the poor intrinsic DNA binding and ATP processing activities of hXRCC2 compared with hRAD51 or RecA (4Takata M. Sasaki M.S. Tachiiri S. Fukushima T. Sonoda E. Schild D. Thompson L.H. Takeda S. Mol. Cell. Biol. 2001; 21: 2858-2866Crossref PubMed Scopus (467) Google Scholar, 42O'Regan P. Wilson C. Townsend S. Thacker J. J. Biol. Chem. 2001; 276: 22148-22153Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and are consistent with studies demonstrating that ATP binding and hydrolysis by hXRCC2 are unnecessary for cellular DNA damage processing (42O'Regan P. Wilson C. Townsend S. Thacker J. J. Biol. Chem. 2001; 276: 22148-22153Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).Fig. 2hXRCC2 modifies ADP-dependent hRAD51·ssDNA complex formation. A, hXRCC2 suppresses hRAD51 ADP-dependent aggregation. The indicated amounts of hXRCC2 and hRAD51 were mixed with 250 nm (nt) ssDNA ([32P]oligo-dT50) with or without ADP (
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