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Biochemical Characterization of the Human RAD51 Protein

2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês

10.1074/jbc.m109915200

ISSN

1083-351X

Autores

Gregory Tombline, Richard Fishel,

Tópico(s)

ATP Synthase and ATPases Research

Resumo

The prototypical bacterial RecA protein promotes recombination/repair by catalyzing strand exchange between homologous DNAs. While the mechanism of strand exchange remains enigmatic, ATP-induced cooperativity between RecA protomers is critical for its function. A human RecA homolog, human RAD51 protein (hRAD51), facilitates eukaryotic recombination/repair, although its ability to hydrolyze ATP and/or promote strand exchange appears distinct from the bacterial RecA. We have quantitatively examined the hRAD51 ATPase. The catalytic efficiency (k cat/K m) of the hRAD51 ATPase was ∼50-fold lower than the RecA ATPase. Altering the ratio of DNA/hRAD51 and including salts that stimulate DNA strand exchange (ammonium sulfate and spermidine) were found to affect the catalytic efficiency of hRAD51. The average site size of hRAD51 was determined to be ∼3 nt (bp) for both single-stranded and double-stranded DNA. Importantly, hRAD51 lacks the magnitude of ATP-induced cooperativity that is a hallmark of RecA. Together, these results suggest that hRAD51 may be unable to coordinate ATP hydrolysis between neighboring protomers. The prototypical bacterial RecA protein promotes recombination/repair by catalyzing strand exchange between homologous DNAs. While the mechanism of strand exchange remains enigmatic, ATP-induced cooperativity between RecA protomers is critical for its function. A human RecA homolog, human RAD51 protein (hRAD51), facilitates eukaryotic recombination/repair, although its ability to hydrolyze ATP and/or promote strand exchange appears distinct from the bacterial RecA. We have quantitatively examined the hRAD51 ATPase. The catalytic efficiency (k cat/K m) of the hRAD51 ATPase was ∼50-fold lower than the RecA ATPase. Altering the ratio of DNA/hRAD51 and including salts that stimulate DNA strand exchange (ammonium sulfate and spermidine) were found to affect the catalytic efficiency of hRAD51. The average site size of hRAD51 was determined to be ∼3 nt (bp) for both single-stranded and double-stranded DNA. Importantly, hRAD51 lacks the magnitude of ATP-induced cooperativity that is a hallmark of RecA. Together, these results suggest that hRAD51 may be unable to coordinate ATP hydrolysis between neighboring protomers. The bacterial RecA ATPase 1The abbreviations used are: ATPaseATP hydrolysis activityhRAD51human RAD51 proteinssDNAsingle-stranded DNAdsDNAdouble-stranded DNAAlF4−aluminum fluorideMES2-(N-morpholino)ethanesulfonic acidntnucleotideRFI and RFIIIreplicative form I and III, respectivelyATPγSadenosine 5′-O- (thiotriphosphate) and its homologs facilitate DNA recombination/repair, although the role of ATP hydrolysis in these processes is not fully understood (1.Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar). All members of the RecA family, including the human RAD51 protein (hRAD51), contain classic Walker A/B motifs, which are fundamentally required for ATP hydrolysis (1.Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar, 2.Brocchieri L. Karlin S. J. Mol. Biol. 1998; 276: 249-264Crossref PubMed Scopus (63) Google Scholar). These motifs are generally conserved among proteins that bind and hydrolyze NTPs (3.Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4437) Google Scholar). RecA proteins mutated at the Walker A/B motifs lack the ability to bind and/or hydrolyze ATP in vitro (4.Kowalczykowski S.C. Biochimie (Paris). 1991; 73: 289-304Crossref PubMed Scopus (83) Google Scholar). While cells expressing these RecA Walker A/B mutants remain viable, they display dramatically reduced levels of recombination and increased radiation sensitivity (5.Konola J.T. Logan K.M. Knight K.L. J. Mol. Biol. 1994; 237: 20-34Crossref PubMed Scopus (54) Google Scholar). ATP hydrolysis activity human RAD51 protein single-stranded DNA double-stranded DNA aluminum fluoride 2-(N-morpholino)ethanesulfonic acid nucleotide replicative form I and III, respectively adenosine 5′-O- (thiotriphosphate) Multiple mitotic and meiotic RecA homologs contribute to eukaryotic recombination/repair. Each of these homologs is likely to have distinct requirements for ATP binding and/or hydrolysis in recombination/repair. For example, four nonessential RecA homologs have been identified inSaccharomyces cerevisiae: RAD51, RAD55, RAD57, and DMC1 (6.Lovett S.T. Mortimer R.K. Genetics. 1987; 116: 547-553Crossref PubMed Google Scholar, 7.Kans J.A. Mortimer R.K. Gene (Amst.). 1991; 105: 139-140Crossref PubMed Scopus (95) Google Scholar, 8.Bishop D.K. Park D. Xu L. Kleckner N. Cell. 1992; 69: 439-456Abstract Full Text PDF PubMed Scopus (999) Google Scholar, 9.Shinohara A. Ogawa H. Ogawa T. Cell. 1992; 69: 457-470Abstract Full Text PDF PubMed Scopus (1071) Google Scholar). Mutation of the Walker A/B motifs of RAD51 and RAD55 results in radiation sensitivity as well as meiotic recombination deficiency (9.Shinohara A. Ogawa H. Ogawa T. Cell. 1992; 69: 457-470Abstract Full Text PDF PubMed Scopus (1071) Google Scholar,10.Johnson R.D. Symington L.S. Mol. Cell. Biol. 1995; 15: 4843-4850Crossref PubMed Scopus (212) Google Scholar). Mutation of the DMC1 Walker A motif results in a dominant meiotic null mutant (11.Dresser M.E. Ewing D.J. Conrad M.N. Dominguez A.M. Barstead R. Jiang H. Kodadek T. Genetics. 1997; 147: 533-544Crossref PubMed Google Scholar). However, similar mutations of RAD57 do not display radiation sensitivity and are only modestly deficient for meiotic recombination (9.Shinohara A. Ogawa H. Ogawa T. Cell. 1992; 69: 457-470Abstract Full Text PDF PubMed Scopus (1071) Google Scholar, 10.Johnson R.D. Symington L.S. Mol. Cell. Biol. 1995; 15: 4843-4850Crossref PubMed Scopus (212) Google Scholar). The complexity of the recombination/repair system is further amplified in higher eukaryotes, since eight vertebrate RecA homologs have been identified (12.Thacker J. Trends Genet. 1999; 15: 166-168Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In contrast to RecA or the yeast homologs, the vertebrate RAD51 gene appears to be required for cellular viability, since Rad51 −/− mice display early embryo lethality and embryo-derived cell lines could not be established (13.Lim D.S. Hasty P. Mol. Cell. Biol. 1996; 16: 7133-7143Crossref PubMed Scopus (634) Google Scholar, 14.Tsuzuki T. Fujii Y. Sakumi K. Tominaga Y. Nakao K. Sekiguchi M. Matsushiro A. Yoshimura Y. Morita T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6236-6240Crossref PubMed Scopus (679) Google Scholar). Similarly, chicken DT40 B-cells lacking endogenous RAD51 were not viable (15.Sonoda E. Sasaki M.S. Buerstedde J.M. Bezzubova O. Shinohara A. Ogawa H. Takata M. Yamaguchiiwai Y. Takeda S. EMBO J. 1998; 17: 598-608Crossref PubMed Scopus (712) Google Scholar). Taken together, these results are consistent with the notion that the RecA homologs of higher eukaryotes are not redundant. Interestingly, the chicken DT40 RAD51-deficient cells could be rescued by the overexpression of an hRAD51 Walker A/B mutant protein that was able to bind but not hydrolyze ATP (16.Morrison C. Shinohara A. Sonoda E. Yamaguchi-Iwai Y. Takata M. Weichselbaum R.R. Takeda S. Mol. Cell. Biol. 1999; 19: 6891-6897Crossref PubMed Scopus (100) Google Scholar). In addition, these rescued cells displayed no increase in radiation sensitivity compared with wild type cells but were less efficient at facilitating recombination-dependent gene targeting at several loci (16.Morrison C. Shinohara A. Sonoda E. Yamaguchi-Iwai Y. Takata M. Weichselbaum R.R. Takeda S. Mol. Cell. Biol. 1999; 19: 6891-6897Crossref PubMed Scopus (100) Google Scholar). This data suggests that the contributions of hRAD51 ATP binding and hydrolysis to viability and recombination/repair may be distinct. Biochemical analysis has proven useful in elucidating the role of ATP hydrolysis in the recombination functions of the RecA protein. It has been suggested that cycles of ATP hydrolysis allow protomers within the RecA nucleoprotein filament to alternate between distinct ATP- and ADP-bound conformational states (1.Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar, 17.Kowalczykowski S.C. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 539-575Crossref PubMed Scopus (250) Google Scholar). The cycling between conformational states appears to drive directional strand exchange during recombination. Historically, these alternating conformations were suspected to facilitate strand exchange by redistributing protomers within the nucleoprotein filament. This assumption was based upon the differential affinities of RecA for DNA in the presence of ATPγS (high affinity) versus in the presence of ADP (low affinity) (1.Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar, 17.Kowalczykowski S.C. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 539-575Crossref PubMed Scopus (250) Google Scholar). An alternative model suggested that strand exchange is facilitated by ATP hydrolysis-dependent rotation of the RecA nucleoprotein filament (18.Cox M.M. Trends Biochem. Sci. 1994; 19: 217-222Abstract Full Text PDF PubMed Scopus (85) Google Scholar). Time lapse electron microscopy of RecA nucleoprotein filaments formed in the presence of ATPγS appeared consistent with this latter proposal (19.Yu X. Jacobs S.A. West S.C. Ogawa T. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8419-8424Crossref PubMed Scopus (210) Google Scholar). These slowly hydrolyzing nucleoprotein filaments remained in an extended yet dynamic state, where the protomers appeared to rotate. The coupling of ATP binding/hydrolysis to recombination by hRAD51 is less certain. In comparison with electron microscopy images of RecA, the hRAD51·ssDNA nucleoprotein filaments formed in the presence of ATPγS appeared less extended, suggesting a diminished response to ATP (20.Benson F.E. Stasiak A. West S.C. EMBO J. 1994; 13: 5764-5771Crossref PubMed Scopus (403) Google Scholar). Yet, ATP was required for hRAD51 to extend the helical pitch (DNA-unwinding) of dsDNA (20.Benson F.E. Stasiak A. West S.C. EMBO J. 1994; 13: 5764-5771Crossref PubMed Scopus (403) Google Scholar) as well as to promote strand exchange between homologous DNA substrates (21.Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 22.Gupta 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, 23.Gupta R.C. Folta-Stogniew E. O'Malley S. Takahashi M. Radding C.M. Mol. Cell. 1999; 4: 705-714Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 24.Gupta R.C. Folta Stogniew E. Radding C.M. J. Biol. Chem. 1999; 274: 1248-1256Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Several recent studies suggest that hRAD51 can be induced to resemble RecA, both structurally and functionally. hRAD51 forms an extended nucleoprotein filament with the transition state mimetic ADP-AlF4− that appears analogous to activated RecA (19.Yu X. Jacobs S.A. West S.C. Ogawa T. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8419-8424Crossref PubMed Scopus (210) Google Scholar). Two additional reports indicated that yeast and human RAD51-mediated DNA strand exchange is greatly enhanced by ammonium sulfate and/or spermidine (25.Sigurdsson 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, 26.Rice 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). The mechanistic basis for ammonium sulfate and spermidine stimulation of RAD51 function is unknown. It is important to note that the rate of RAD51 strand exchange remains 3–5-fold lower than RecA (25.Sigurdsson 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, 26.Rice 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). Furthermore, RAD51 converts a maximum of 30–60% of ssDNA to form II products in 60 min, whereas RecA converts 100% ssDNA to form II in 15 min (25.Sigurdsson 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, 26.Rice 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). Strand exchange by RecA has been shown to be largely independent of ATP hydrolysis (27.Menetski J.P. Bear D.G. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 21-25Crossref PubMed Scopus (235) Google Scholar, 28.Rehrauer W.M. Kowalczykowski S.C. J. Biol. Chem. 1993; 268: 1292-1297Abstract Full Text PDF PubMed Google Scholar, 29.Kowalczykowski S.C. Krupp R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3478-3482Crossref PubMed Scopus (106) Google Scholar, 30.Shan Q. Cox M.M. Inman R.B. J. Biol. Chem. 1996; 271: 5712-5724Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). However, RecA-dependent bypass of heterologous DNA absolutely requires ATP hydrolysis (30.Shan Q. Cox M.M. Inman R.B. J. Biol. Chem. 1996; 271: 5712-5724Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 31.Rosselli W. Stasiak A. EMBO J. 1991; 10: 4391-4396Crossref PubMed Scopus (86) Google Scholar, 32.Kim J.I. Cox M.M. Inman R.B. J. Biol. Chem. 1992; 267: 16438-16443Abstract Full Text PDF PubMed Google Scholar). ATP hydrolysis-dependent bypass of heterologous DNA has been argued to require the generation of rotory torque and/or recycling of the RecA protomers during the strand exchange reaction (30.Shan Q. Cox M.M. Inman R.B. J. Biol. Chem. 1996; 271: 5712-5724Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 33.Shan Q. Cox M.M. J. Biol. Chem. 1997; 272: 11063-11073Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 34.MacFarland K.J. Shan Q. Inman R.B. Cox M.M. J. Biol. Chem. 1997; 272: 17675-17685Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). RAD51 displays a dramatically reduced capacity to bypass heterologous DNA during strand exchange (26.Rice 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, 35.Holmes V.F. Benjamin K.R. Crisona N.J. Cozzarelli N.R. Nucleic Acids Res. 2001; 29: 5052-5057Crossref PubMed Scopus (24) Google Scholar, 36.Sung P. Robberson D.L. Cell. 1995; 82: 453-461Abstract Full Text PDF PubMed Scopus (434) Google Scholar, 37.Namsaraev E.A. Berg P. J. Biol. Chem. 2000; 275: 3970-3976Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Taken together, these observations suggest that altered ATP processing might account for the disparities between hRAD51 and bacterial RecA activities. To understand the ATP-dependent recombination functions of hRAD51, we have performed the first quantitative examination of the hRAD51 ATPase. Our results allow a detailed comparison with the bacterial RecA protein, the only other homolog in this family in which similar studies have been performed. Of particular importance is our observation that hRAD51 appears to lack the magnitude of ATP-induced cooperativity displayed by RecA. Our results suggest that other regulatory factors or RecA homologs may be required to assemble and control an hRAD51 nucleoprotein filament. Chemicals of the highest grade were obtained from Amresco (Solon, OH) or Sigma. Phosphoric/sulfuric acid-washed charcoal was obtained from Sigma (catalog no. C-5510). ATP was purchased from Amersham Biosciences, dissolved in water, and adjusted for pH. ATP concentration was determined by absorbance at 260 nm with ε = 1.54 × 104. [γ-32P]ATP was purchased from PerkinElmer Life Sciences. Bacteriophage φX174 DNA was purchased from New England Biolabs (Beverly, MA). Linear, blunt-ended dsDNA (RFIII) was prepared by treatment of bacteriophage φX174 dsDNA (RFI) with endonuclease StuI (New England Biolabs) followed by phenol extraction and ethanol precipitation. The DNA was resuspended in 10 mm Tris-HCl (pH 8), 1 mm EDTA and analyzed by agarose gel electrophoresis. The concentration of DNA is expressed as mol of nt (ssDNA) or bp (dsDNA). hRAD51 was purified as previously described (38.Baumann P. Benson F.E. Hajibagheri N. West S.C. Mutat. Res. DNA Repair. 1997; 384: 65-72Crossref PubMed Scopus (63) Google Scholar) with several modifications. Briefly, hRAD51 cDNA was subcloned into pET24d (Novagen), and induction was performed in theE. coli strain BLR21pLysS. Cells were lysed by three freeze/thaw cycles and centrifuged at ∼160,000 × gfor 1 h. The supernatant was dialyzed overnight against 4 liters of 100 mm Tris acetate (pH 7.5), 5% glycerol, and 7 mm spermidine HCl. The hRAD51 precipitate was collected by centrifugation and resuspended (∼10 mg/ml) in P buffer (100 mm potassium phosphate (pH 7.0), 10% glycerol, 150 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol) (fraction I). Fraction I was separated by chromatography through Reactive Blue-4-agarose (Sigma). Protein was eluted with a 750 mm step of NaCl and dialyzed overnight against 4 liters of H buffer (20 mm HEPES (pH 7.5), 150 mm NaCl, 10% glycerol, 1 mm EDTA, 1 mm dithiothreitol) (fraction II). Fraction II was loaded onto a heparin-Sepharose CL-6B column and eluted with a 750 mm NaCl step (fraction III). Fraction III was dialyzed overnight against 4 liters of H buffer (fraction IV), loaded onto a Mono-Q column (HR5/5 prepacked from Amersham Biosciences), and eluted with a gradient of 150–750 mm NaCl over 20 ml (fraction V). hRAD51 was dialyzed twice against 2 liters of modified H buffer (containing 0.1 mm EDTA) and was stored on ice. Occasionally, an additional purification step was necessary to remove trace contaminants. In this case, the protein was dialyzed against modified P buffer (P buffer containing 100 mm NaCl) after Mono-Q, chromatographed on a Mono-S column equilibrated with modified P buffer, and eluted with a 15-ml gradient of 100–750 mmNaCl. This was followed by dialysis against modified H buffer (0.1 mm EDTA). Purified hRAD51 can be stored in modified H buffer at 0 or −80 °C for several months without appreciable loss of ATPase activity. Protein concentration was determined by amino acid analysis (Keck Facility, Yale University). Preparations were found to be nuclease-free by incubation with both single- and double-stranded phage DNA as well as small oligonucleotides. Unless otherwise indicated, the 10-μl reactions contained 1 μm hRAD51 and 6 μmDNA (nt or bp). All reactions were performed in an A buffer (20 mm HEPES, pH 7.5, 150 mm NaCl, 10% glycerol, 1 mm dithiothreitol, 2 mm magnesium acetate). In addition to the indicated amounts of ATP, 1 μCi of γ-[32P]ATP was included in each reaction. To achieve consistency in such small reactions, mixes containing either DNA/ATP or hRAD51 were added separately using a repeat pipettor (Eppendorf). Reactions were initiated by adding the hRAD51 mix last and incubated at 37 °C for 30–60 min in an Ericomp thermal cycler with a heated lid to prevent evaporation. 400 μl of ice cold 10% activated charcoal (Norit) in 10 mm EDTA was added to terminate the reactions. Tubes were vortexed and placed on ice for a minimum of 3 h to allow maximal binding of free ATP to the charcoal. After centrifugation for 30 min, two 50-μl aliquots were counted by the Cerenkov method. hRAD51 was omitted from two additional reactions, which contained either the lowest or highest amount of ATP. After processing with Norit, background hydrolysis determined for these reactions was averaged and subtracted from reactions containing hRAD51. In general, there was no significant difference in background between lowest and highest ATP reactions. Total counts in each reaction were determined by the Cerenkov method for duplicate reactions that had not been processed with Norit. The average of at least three such duplicates was taken for the total cpm/reaction. Specific activity of ATP (cpm/mol; [γ-32P]ATP/ATP) in each was calculated by dividing the total counts in each reaction by the amount of total ATP. The amount of ATP hydrolyzed (mol) in each reaction was determined by dividing the adjusted hydrolysis value (cpm) by the specific activity of ATP in each reaction. Molar ratios were varied by adjustment of the DNA or hRAD51 concentrations in otherwise identical reactions. In all reactions, conditions were normalized for the DNA or hRAD51 storage buffers. The pH was varied using similar reaction buffers except that MES was used for the range between pH 6.2 and pH 7.2, and HEPES was varied between pH 7.2 and 8.2. For ATPase reactions containing additional salts, 0.5 μl of 2 m (NH4)2SO4(pH 8.0) and/or 0.5 μl of 80 mm spermidine HCl were added to reactions with a final volume of 10 μl, yielding final concentrations of 100 mm and/or 4 mm, respectively. These reactions were processed as above. Using a modification of a previously published method (38.Baumann P. Benson F.E. Hajibagheri N. West S.C. Mutat. Res. DNA Repair. 1997; 384: 65-72Crossref PubMed Scopus (63) Google Scholar), we purified hRAD51 to near homogeneity (Fig. 1). The ATPase activity of hRAD51 was measured by the Norit method (see “Experimental Procedures”), and the unique conversion of ATP to ADP was confirmed by thin layer chromatography (TLC) (39.Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). We found the Norit method to be superior to TLC or PAGE analysis because of the ease of data acquisition, reduced expense, and the large number of analyses that could be performed in tandem. Past experience has suggested that the separation efficiency of Norit exceeds 90% and can be increased by prolonged incubation of the terminated reactions at 0 °C (39.Gradia S. Acharya S. Fishel R. Cell. 1997; 91: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar,40.Adzuma K. Mizuuchi K. J. Biol. Chem. 1991; 266: 6159-6167Abstract Full Text PDF PubMed Google Scholar). While a previous report indicated that ATP hydrolysis by hRAD51 was very inefficient (k cat(ssDNA) = 0.2 min−1, k cat(dsDNA) = 0.1 min−1, k cat = 0.03 min−1), these values were based on a single ATP concentration (200 μm) (21.Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar). To confirm and extend these studies and to examine the rate-limiting step(s) within the hydrolysis cycle, we performed classic Michealis-Menten analysis to define theK m and k cat(V max/[hRAD51]o). We determined these values for the hRAD51 ATPase activity in the presence of ssDNA, supercoiled dsDNA (RFI), linear dsDNA (RFIII), and in the absence of DNA. The k cat values (k cat(ssDNA) = 0.21 min−1;k cat(dsRFI) = 0.1 min−1;k cat(dsRFIII) = 0.07 min−1;k cat = 0.07 min−1; Fig. 2, A and B, and Table I) agreed with values previously reported (21.Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar). These data indicate that thek cat(ssDNA) for hRAD51 is ∼150-fold lower than bacterial RecA (k cat(ssDNA) = 28 min−1), and the k cat(dsDNA) is ∼220-fold lower than bacterial RecA (k cat(dsDNA) = 22 min−1 at pH 6.2) (41.Weinstock G.M. McEntee K. Lehman I.R. J. Biol. Chem. 1981; 256: 8845-8849Abstract Full Text PDF PubMed Google Scholar, 42.Weinstock G.M. McEntee K. Lehman I.R. J. Biol. Chem. 1981; 256: 8829-8834Abstract Full Text PDF PubMed Google Scholar, 43.Pugh B.F. Cox M.M. J. Biol. Chem. 1987; 262: 1326-1336Abstract Full Text PDF PubMed Google Scholar). The k cat in the absence of DNA is ∼4-fold higher than the bacterial RecA (k cat = 0.015 min−1) (41.Weinstock G.M. McEntee K. Lehman I.R. J. Biol. Chem. 1981; 256: 8845-8849Abstract Full Text PDF PubMed Google Scholar, 42.Weinstock G.M. McEntee K. Lehman I.R. J. Biol. Chem. 1981; 256: 8829-8834Abstract Full Text PDF PubMed Google Scholar, 43.Pugh B.F. Cox M.M. J. Biol. Chem. 1987; 262: 1326-1336Abstract Full Text PDF PubMed Google Scholar). In addition, hRAD51 displays an equal or higher apparent affinity for ATP (K m(ssDNA) = 23 ± 3 μm; K m(dsDNA(RFI)) = 27 ± 4 μm;K m(dsDNA(RFIII)) = 26 ± 5 μm; K m = 110 ± 22 μm) compared with bacterial RecA (S0.5(ssDNA)≈ 20–60 μm; S0.5(dsDNA(RFI)) ≈ 100 μm at pH 6.2; S0.5 ≈ 100 μm) (17.Kowalczykowski S.C. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 539-575Crossref PubMed Scopus (250) Google Scholar, 41.Weinstock G.M. McEntee K. Lehman I.R. J. Biol. Chem. 1981; 256: 8845-8849Abstract Full Text PDF PubMed Google Scholar), the substrate concentration where half-maximal activity occurs is S0.5 in a cooperative system and equalsK m in a noncooperative system (44.Segel I.H. Biochemical Calculations. John Wiley and Sons, Inc., New York1976: 309-317Google Scholar). In general, DNA appears to decrease the hRAD51 K m equivalently (Fig. 2, A and B, and Table I). Calculation of the hRAD51 ATPase catalytic efficiency (k cat/K m) suggests that ssDNA (150 s−1m−1) induces the ATPase no more than 2–3-fold more than dsDNA (∼68 s−1m−1) and at least 5-fold more than in the absence of DNA (11 s−1m−1). The catalytic efficiency of hRAD51 is ∼50-fold less than bacterial RecA (8300 s−1m−1).Table ISummary of hRAD51 ATPase data (see Figs. 2 and 5)ConditionK mV maxk catk cat/K mn Hμmμm · min−1min−1s−1 · m−1ssDNA23 ± 30.211 ± 0.0050.211500.79dsDNA (RFI)27 ± 40.114 ± 0.0030.11680.76dsDNA (RFIII)26 ± 50.071 ± 0.0020.07450.74No DNA110 ± 220.073 ± 0.0060.07110.701.5M NaCl16.9 ± 20.197 ± 0.0060.201900.81 Open table in a new tab A commonly used method for determining ATP-induced cooperativity is by calculating the Hill coefficient (44.Segel I.H. Biochemical Calculations. John Wiley and Sons, Inc., New York1976: 309-317Google Scholar). The Hill coefficient was originally developed for the analysis of cooperative fractional saturation for a ligand binding to multiple interdependent sites (45.Dahlquist F.W. Methods Enzymol. 1978; 48: 270-299Crossref PubMed Scopus (298) Google Scholar). If one assumes that the rate of an enzymatic reaction is proportional to the fractional saturation of the enzyme, then a slope (Hill coefficient) greater than one derived from a plot of the fractional rate [v/(V max− v)] versus the substrate concentration ([S]) indicates positive cooperativity (44.Segel I.H. Biochemical Calculations. John Wiley and Sons, Inc., New York1976: 309-317Google Scholar). In general, the largest value for cooperativity occurs at half-maximal fractional saturation. As the fractional saturation approaches unity, the Hill coefficient also approaches 1. This appears to be the case for the RecA ATPase (41.Weinstock G.M. McEntee K. Lehman I.R. J. Biol. Chem. 1981; 256: 8845-8849Abstract Full Text PDF PubMed Google Scholar,46.Mikawa T. Masui R. Kuramitsu S. J. Biochem. 1998; 123: 450-457Crossref PubMed Scopus (21) Google Scholar). The Hill coefficient (n H) of RecA varies from n H = 3 to n H = 11 at ATP concentrations below or slightly above the S0.5. At ATP concentrations above the S0.5 (>100 μm), ATP-induced cooperativity becomes less apparent, since the Hill coefficient equals 1. Three methods of analysis of hRAD51 ATPase data indicate that hRAD51 lacks ATP-induced cooperativity. First, in the absence or presence of DNA, the rate of ATP hydrolysis was easily fit to a simple hyperbolic curve (the Michaelis-Menten equation; Fig. 2 A). Second, the slope of the same ATPase data plotted by the double-reciprocal method is linear (not concave upward; Fig. 2 B). Third, the slope of hRAD51 ATPase data plotted as a fractional rate versus ATP concentration (Hill coefficient) was ∼1 in all conditions (ssDNAn H = 0.79; dsDNA (RFI) n H= 0.76; dsDNA (RFIII) n H = 0.74; absence of DNAn H = 0.70; Fig. 2 C and Table I). hRAD51 also failed to display ATP-induced cooperativity in the range of ATP concentrations below the K m (Fig. 3). These data contrast with the cooperativity shown by RecA. RecA can efficiently utilize dsDNA as a cofactor for hydrolysis at pH 6.2. However, at pH 8.0, the dsDNA-dependent ATPase can only be measured after a lag of several hours (1.Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar, 17.Kowalczykowski S.C. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 539-575Crossref PubMed Scopus (250) Google Scholar, 42.Weinstock G.M. McEntee K. Lehman I.R. J. Biol. Chem. 1981; 256: 8829-8834Abstract Full Text PDF PubMed Google Scholar). This lag at higher pH is absent when the carboxyl-terminal domain of bacterial RecA is deleted or when purified unwound dsDNA (form X) is used as a cofactor (43.Pugh B.F. Cox M.M. J. Biol. Chem. 1987; 262: 1326-1336Abstract Full Text PDF PubMed Google Scholar, 47.Benedict R.C. Kowalczykowski S.C. J. Biol. Chem. 1988; 263: 15513-15520Abstract Full Text PDF PubMed Google Scholar, 48.Tateishi S. Horii T. Ogawa T. Ogawa H. J. Mol. Biol. 1992; 223: 115-129Crossref PubMed Scopus (55) Google Scholar). We examined the pH dependence of the hRAD51 ATPase between 6.2 and 8.2 and found no difference in ATPase activity for either ssDNA or dsDNA (RFIII) (Fig. 4, A andB, respectively). It is interesting to note that a comparison of the hRAD51 and RecA sequences suggests that the carboxyl-terminal domain is missing in hRAD51 (1.Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar, 2.Brocchieri L. Karlin S. J. Mol. Biol. 1998; 276: 249-264Crossref PubMed Scopus (63)

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