Homologous DNA Pairing by Human Recombination Factors Rad51 and Rad54

Human Rad51 (hRad51) and Rad54 proteins are key members of the RAD52 group required for homologous recombination. We show an ability of hRad54 to promote transient separation of the strands in duplex DNA via its ATP hydrolysis-driven DNA supercoiling function. The ATPase, DNA supercoiling, and DNA strand opening activities of hRad54 are greatly stimulated through an interaction with hRad51. Importantly, we demonstrate that hRad51 and hRad54 functionally cooperate in the homologous DNA pairing reaction that forms recombination DNA intermediates. Our results should provide a biochemical model for dissecting the role of hRad51 and hRad54 in recombination reactions in human cells. Human Rad51 (hRad51) and Rad54 proteins are key members of the RAD52 group required for homologous recombination. We show an ability of hRad54 to promote transient separation of the strands in duplex DNA via its ATP hydrolysis-driven DNA supercoiling function. The ATPase, DNA supercoiling, and DNA strand opening activities of hRad54 are greatly stimulated through an interaction with hRad51. Importantly, we demonstrate that hRad51 and hRad54 functionally cooperate in the homologous DNA pairing reaction that forms recombination DNA intermediates. Our results should provide a biochemical model for dissecting the role of hRad51 and hRad54 in recombination reactions in human cells. human Rad51 yeast Rad51 single-stranded bovine serum albumin adenosine 5′-(β,γ-imino)triphosphate adenosine 5′-3-O-(thio)triphosphate In eukaryotic organisms, the repair of DNA double-stranded breaks by homologous recombination is mediated by a group of evolutionarily conserved genes known as the RAD52 epistasis group. Members of the RAD52 group (RAD51, RAD52,RAD54, RAD55, RAD57, RAD59, and RDH54/TID1) were first uncovered in genetic screens in the budding yeast Saccharomyces cerevisiae (1Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar,2Sung P. Trujillo K. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (158) Google Scholar). In mammals, the efficiency of homology-directed recombinational DNA repair is modulated by the tumor suppressors BRCA1 and BRCA2 (3Pierce A.J. Stark J.M. Araujo F.D. Moynahan M.E. Berwick M. Jasin M. Trends Cell Biol. 2001; 11: S52-S59Abstract Full Text PDF PubMed Scopus (235) Google Scholar), providing a compelling link between this DNA repair pathway and the suppression of tumor formation. The involvement of the homologous recombination machinery in the maintenance of genome stability and tumor suppression underscores the need for deciphering the action mechanism of this machinery. During the recombinational repair of DNA double-stranded breaks, a single-stranded DNA intermediate is utilized by the recombination machinery to invade a DNA homolog, most often the sister chromatid, to form a DNA joint molecule referred to as a d-loop (2Sung P. Trujillo K. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (158) Google Scholar).d-Loop formation is critical for subsequent steps in the recombination reaction, which include repair DNA synthesis and resolution of recombination intermediates (1Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar, 2Sung P. Trujillo K. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (158) Google Scholar), that lead to the restoration of the integrity of the injured chromosome. In the past several years, biochemical studies have begun to shed light on the functions of the human RAD52 group proteins in DNA joint formation. Much of the published work has centered on the human Rad51 (hRad51)1 protein, which is structurally related to the Escherichia colirecombinase enzyme RecA (4Shinohara A. Ogawa H. Matsuda Y. Ushio N. Ikeo K. Ogawa T. Nat. Genet. 1993; 4: 239-243Crossref PubMed Scopus (30) Google Scholar). Like RecA, hRad51 assembles into a right-handed filament on single-stranded (ss) DNA in a reaction that is dependent on ATP binding (reviewed in Ref. 5Yu X. Jacobs S.A. West S.C. Ogawa T. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 17: 8419-8424Crossref Scopus (205) Google Scholar). Importantly, hRad51 protein has been shown to have DNA pairing and strand exchange activities that yield DNA joints between homologous ssDNA and double-stranded DNA substrates (6Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 7Gupta R.C. Bazemore L.R. Golub E.I. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 463-468Crossref PubMed Scopus (238) Google Scholar, 8Sigurdsson S. Trujillo K. Song B.-W. Stratton S. Sung P. J. Biol. Chem. 2001; 276: 8798-8806Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The homologous pairing and strand exchange function of hRad51 is augmented by replication protein A (RPA), a heterotrimeric single-stranded DNA binding factor (6Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 8Sigurdsson S. Trujillo K. Song B.-W. Stratton S. Sung P. J. Biol. Chem. 2001; 276: 8798-8806Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), by hRad52 protein (9Benson F.E. Baumann P. West S.C. Nature. 1998; 391: 401-404Crossref PubMed Scopus (331) Google Scholar), and by the Rad51B-Rad51C heterodimeric complex (10Sigurdsson S. Van Komen S. Bussen W. Schild D. Albala J.S. Sung P. Genes Dev. 2001; 15: 3308-3318Crossref PubMed Scopus (185) Google Scholar), which is the functional equivalent of the yeast Rad55-Rad57 complex (11Sung P. Genes Dev. 1997; 11: 1111-1121Crossref PubMed Scopus (461) Google Scholar). The RAD54 encoded product belongs to the Swi2/Snf2 protein family (12Eisen J.A. Sweder K.S. Hanawalt P.C. Nucleic Acids Res. 1995; 23: 2715-2723Crossref PubMed Scopus (618) Google Scholar). Purified hRad54 exhibits DNA-dependent ATPase and DNA supercoiling activities (13Swagemakers S.M.A. Essers J. de Wit J. Hoeijmakers J.H.J. Kanaar R. J. Biol. Chem. 1998; 273: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 14Tan T.L. Essers J. Citterio E. Swagemakers S.M. de Wit J. Benson F.E. Hoeijmakers J.H. Kanaar R. Curr. Biol. 1999; 9: 325-328Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 15Ristic D. Wyman C. Paulusma C. Kanaar R. Proc. Natl. Acad. Sci. U. S. A. 2001; 84: 8454-8460Crossref Scopus (110) Google Scholar). However, the manner in which hRad54 influences the hRad51-mediated recombination reaction has remained mysterious. Here we report our biochemical studies that show functional interactions between hRad51 and hRad54 in DNA supercoiling and homologous DNA pairing reactions. We discuss how hRad51 and hRad54 cooperate to make DNA joints during recombination processes. The first 238 amino acid residues of the human Rad54 protein were fused to glutathioneS-transferase in the vector pGEX-3X. The fusion protein was expressed in E. coli strain BL21 (DE3) and purified from inclusion bodies by preparative denaturing polyacrylamide gel electrophoresis and used as antigen for raising polyclonal antibodies in rabbits. The same antigen was covalently conjugated to cyanogen bromide-activated Sepharose 4B and used as affinity matrix to purify the antibodies from rabbit antisera, as described (16Sung P. Prakash L. Matson S.W. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8951-8955Crossref PubMed Scopus (167) Google Scholar). A recombinant baculovirus containing the cloned hRad54 cDNA with an added FLAG epitope at the C terminus was generated. HighFive insect cells were infected with the recombinant baculovirus at a multiplicity of infection of 10 and harvested after 48 h of incubation. An extract was prepared from 500 ml of insect cell culture (5 × 108 cells) using a French Press in 60 ml of cell breakage buffer (50 mm Tris-HCl, pH 7.5, 2 mm EDTA, 10% sucrose, 200 mm KCl, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and the following protease inhibitors at 3 μg/ml each: aprotinin, chymostatin, leupeptin, and pepstatin). After centrifugation (100,000 × g for 60 min), the clarified extract was loaded onto a Q-Sepharose column (10-ml matrix). The flow-through fraction from the Q column was fractionated in a sulfopropyl-Sepharose column (10-ml matrix) with a 50-ml, 0–700 mm KCl gradient in K buffer (20 mmKH2PO4 at pH 7.4, 0.5 mm EDTA, 1 mm dithiothreitol, and 10% glycerol). Fractions containing the peak of hRad54 were pooled and loaded onto a 1-ml Macro-hydroxyapatite (Bio-Rad) column, which was eluted with 30 ml of 0–300 mm KH2PO4 in K buffer. The peak fractions were pooled and mixed with 1.5 ml of Anti-FLAG M2 agarose (Sigma) and rocked for 3 h at 4 °C. The FLAG agarose was washed three times with 3 ml of 150 mm KCl in buffer K before eluting hRad54 using the same buffer containing 1 mg/ml of the FLAG peptide (Sigma). hRad54 (∼1 mg) eluted from the FLAG matrix was concentrated in a Centricon-30 microconcentrator to 5 mg/ml and stored in small aliquots at −70 °C. The hRad51 protein was expressed in the E. coli RecA-deficient strain BLR (DE3) and purified to near homogeneity using our previously described procedure (8Sigurdsson S. Trujillo K. Song B.-W. Stratton S. Sung P. J. Biol. Chem. 2001; 276: 8798-8806Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The hrad51 K133R mutant was expressed and purified to near homogeneity in exactly the same way. E. coli topoisomerase I was purified to near homogeneity from the E. coli strain JM101 with plasmid pJW312-sal containing the topA gene under the Lac promoter, as described (17Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Crossref PubMed Scopus (59) Google Scholar). Purified hRad51 and bovine serum albumin (BSA) were coupled to Affi-Gel 15 beads at 4 °C following the instructions of the manufacturer (Bio-Rad). The resulting matrices contained 4 and 12 mg/ml hRad51 and BSA, respectively. Purified hRad54 (1.2 μg) was mixed with 5 μl of Affi-Rad51 or Affi-BSA at 4 °C for 30 min in 30 μl of buffer containing 100 mm KCl and 0.1% Triton X-100 by constant tapping. The beads were washed twice with 50 μl of the same buffer before being treated with 30 μl of 2% SDS at 37 °C for 5 min to elute the bound hRad54. The various fractions (4 μl each) were analyzed by immunoblotting to determine their content of hRad54. Topologically relaxed φX174 DNA was prepared as described (18Van Komen S. Petukhova G. Sigurdsson S. Stratton S. Sung P. Mol. Cell. 2000; 6: 563-572Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), and pBluescript SK DNA was made in E. coli DH5α and purified as described (19Petukhova G. Stratton S. Sung P. Nature. 1998; 393: 91-94Crossref PubMed Scopus (345) Google Scholar). The oligonucleotide used in the d-loop reaction is complementary to positions 1932–2022 of the pBluescript SK DNA and had the sequence 5′-AAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT-3′. This oligonucleotide was 5′ end-labeled with [γ-32P]ATP and T4 polynucleotide kinase. The hRad54 protein (60 nm) was incubated with replicative form I φX174 DNA (30 μm base pairs) and 1.5 mm [γ-32P]ATP with or without 400 nm hRad51 or yRad51 in 10 μl of reaction buffer (20 mm Tris-HCl, pH 7.4, 25 mm KCl, 1 mm dithiothreitol, 4 mm MgCl2, 100 μg/ml BSA) at 30 °C for the indicated times. The level of ATP hydrolysis was determined by thin layer chromatography, as described (19Petukhova G. Stratton S. Sung P. Nature. 1998; 393: 91-94Crossref PubMed Scopus (345) Google Scholar). Increasing amounts of hRad54 were incubated with 80 ng of relaxed φX174 DNA (12 μm nucleotides) for 2 min at 23 °C in 12 μl of reaction buffer (20 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 1 mmdithiothreitol, 100 μm ATP, and an ATP-regenerating system consisting of 10 mm creatine phosphatase and 28 μg/ml creatine kinase). Following the addition of 100 ng of E. coli topoisomerase I in 0.5 μl, the reactions were incubated for 10 min at 23 °C and then deproteinized by treatment with 0.5% SDS and proteinase K (0.5 mg/ml) for 10 min at 37 °C. Samples were run on agarose in buffer mm pH 7.4, 0.5 mm at 23 °C and then with In the in 4 the relaxed DNA was incubated with the indicated amounts of hRad51 and hRad54 for 2 min at 23 by the addition of topoisomerase and a at 23 °C. the in C and 4 the reactions were in the same manner that of was used of The DNA were in a agarose gel containing 10 μm in DNA and DNA strand for supercoiling, as D. Wyman C. Paulusma C. Kanaar R. Proc. Natl. Acad. Sci. U. S. A. 2001; 84: 8454-8460Crossref Scopus (110) Google and Van Komen (18Van Komen S. Petukhova G. Sigurdsson S. Stratton S. Sung P. Mol. Cell. 2000; 6: 563-572Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The from ATP hydrolysis the of a hRad54 on a of protein and a amounts of hRad54 and nm in were incubated with relaxed DNA (20 μm nucleotides) coli topoisomerase I in buffer that contained The of hRad54 nm) was incubated with the DNA in the of topoisomerase and in the of topoisomerase with the of ATP or the of ATP by and DNA or DNA incubated with topoisomerase was The reaction were run in a agarose which was treated with to the DNA amounts of hRad54 and nm in were incubated with relaxed DNA (20 μm nucleotides) and in buffer that contained The of hRad54 nm) was incubated with the DNA in the of and in the of with the of ATP or the of ATP by and DNA and DNA incubated with in the of hRad54 were The reaction were run in a agarose gel containing 10 μm the reactions μl, in hRad51 or hrad51 K133R nm) was incubated with the oligonucleotide μm nucleotides) for 3 min at 37 °C in μl of reaction buffer (20 mm Tris-HCl, pH 7.4, 100 μg/ml BSA, 1.5 mm MgCl2, 2 mm ATP, and the system described This was by the addition of hRad54 nm) in 1 μl and at 23 °C for 2 The reaction was by the pBluescript SK replicative form DNA μm base pairs) in 2 The reaction was incubated at 30 and aliquots were at the indicated and run in agarose in buffer. The were and the of d-loop were by The reactions in which ATP, or hRad54 was or ATP was by or were to a were and in exactly the same Human Rad51 was expressed in a coli strain and purified to near homogeneity as described previously (8Sigurdsson S. Trujillo K. Song B.-W. Stratton S. Sung P. J. Biol. Chem. 2001; 276: 8798-8806Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The hrad51 K133R which the of the conserved in the A binding to was expressed and In with previously published results C. A. E. Y. M. S. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar), hrad51 K133R has ATPase with hRad51 We cloned the human RAD54 cDNA from a cDNA using the The cDNA was to that with the published sequence R. C. Swagemakers S.M. Essers J. J.H. A. J.M. Hoeijmakers J.H. Curr. Biol. 1996; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). We hRad54 protein with a FLAG epitope at the terminus and expressed in insect cells by the of a recombinant baculovirus We of hRad54 1 from 500 ml of insect cell culture by a of column and affinity binding to an for the FLAG The purified hRad54 has a level of DNA-dependent ATPase to that described in the (13Swagemakers S.M.A. Essers J. de Wit J. Hoeijmakers J.H.J. Kanaar R. J. Biol. Chem. 1998; 273: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (111) Google Golub E.I. R.C. Radding C.M. Nucleic Acids Res. 1997; PubMed Scopus Google that the amino terminus of hRad54 hRad51 in in and in the yeast However, in of and treatment of cells with a DNA T.L. Essers J. Citterio E. Swagemakers S.M. de Wit J. Benson F.E. Hoeijmakers J.H. Kanaar R. Curr. Biol. 1999; 9: 325-328Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). purified hRad54 with we coupled hRad51 to Affi-Gel beads to as affinity matrix for binding hRad54. shown in purified hRad54 was on beads on that contained a purified hRad54 fraction was the protein bound to the beads The results a and interaction between hRad51 and hRad54. T.L. Essers J. Citterio E. Swagemakers S.M. de Wit J. Benson F.E. Hoeijmakers J.H. Kanaar R. Curr. Biol. 1999; 9: 325-328Abstract Full Text Full Text PDF PubMed Scopus (181) Google an ability of hRad54 to the DNA of a plasmid in the of DNA The of DNA was dependent on ATP hydrolysis by hRad54 T.L. Essers J. Citterio E. Swagemakers S.M. de Wit J. Benson F.E. Hoeijmakers J.H. Kanaar R. Curr. Biol. 1999; 9: 325-328Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). The same group used to provide that hRad54 on DNA ATP is D. Wyman C. Paulusma C. Kanaar R. Proc. Natl. Acad. Sci. U. S. A. 2001; 84: 8454-8460Crossref Scopus (110) Google Scholar). A the for DNA supercoiling by hRad54 is in A. The protein on DNA as a and in the DNA of the by treatment with E. coli topoisomerase I to the of and the formation of an DNA (18Van Komen S. Petukhova G. Sigurdsson S. Stratton S. Sung P. Mol. Cell. 2000; 6: 563-572Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Here we used the same to the ability of hRad54 to shown in 3 in the of purified hRad54 protein a in the DNA (18Van Komen S. Petukhova G. Sigurdsson S. Stratton S. Sung P. Mol. Cell. 2000; 6: 563-572Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The DNA supercoiling reaction is dependent on ATP as by its or with a or 3 We the as a of hRad54 on the DNA D. Wyman C. Paulusma C. Kanaar R. Proc. Natl. Acad. Sci. U. S. A. 2001; 84: 8454-8460Crossref Scopus (110) Google A to transient DNA strand opening by the of a relaxed DNA to the as Van Komen (18Van Komen S. Petukhova G. Sigurdsson S. Stratton S. Sung P. Mol. Cell. 2000; 6: 563-572Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). C that of relaxed DNA with hRad54 the relaxed DNA to as indicated by the of and DNA The DNA strand opening reaction is dependent on ATP hydrolysis 3 The results and E.I. R.C. Radding C.M. Nucleic Acids Res. 1997; PubMed Scopus Google have a interaction between hRad51 and hRad54. We the hRad54 ATPase interaction with hRad51. shown in a of ATP hydrolysis was hRad54 was with hRad51. The that yRad51 was in this reaction 4 that the action of hRad51 is hRad51 is known to have a ATPase F.E. A. West S.C. J. PubMed Scopus Google Scholar), the that the hrad51 K133R mutant protein, which ATP C. A. E. Y. M. S. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar), was as in ATP hydrolysis indicated that the in ATP hydrolysis was of of the hRad54 ATPase function. We the DNA supercoiling of hRad54 by hRad51. The results that hRad51 the supercoiling reaction, as indicated by a level of DNA 4 supercoiling by hRad54 to DNA strand opening we that hRad51 promote this the inclusion of hRad51 greatly the of the relaxed DNA by 4 with the inclusion of DNA or of DNA was ATP was or by the and and the results that hRad51 the ability of Rad54 to DNA and DNA The hrad51 K133R protein was as as hRad51 in the DNA supercoiling and strand opening activities of hRad54 we that yRad51 the hRad54 activities a of in the hRad51 The of is to recombination by of their ability to the homologous DNA pairing reaction that DNA joints (2Sung P. Trujillo K. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (158) Google Scholar, S.C. 1998; Scholar). hRad51 and hRad54 E.I. R.C. Radding C.M. Nucleic Acids Res. 1997; PubMed Scopus Google and hRad51 the various activities of hRad54 was of to the of hRad54 on hRad51-mediated homologous DNA The homologous pairing the of single-stranded oligonucleotide into a homologous to before E. Sung P. S.C. J. 2000; PubMed Scopus (135) Google and 5 hRad51 by is at Importantly, the inclusion of hRad54 d-loop formation formation by the of hRad51 and hRad54 ATP d-loop was ATP was or was by or 5 the a of formation and of that level its by 1 min and In by the reaction of or d-loop remained and a synthesis and to a for the of T. T. M. T. J. Biol. Chem. Scholar, Van E. A. West S.C. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). the filament ATP hydrolysis S.C. 1998; Scholar), we the that the 5 related to ATP of hRad51. this we used the hrad51 K133R mutant protein, which ATP C. A. E. Y. M. S. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar), with hRad54 in the d-loop to with hrad51 the d-loop with a level hRad51 was used by 4 of the ssDNA or of the pBluescript plasmid DNA had been into the d-loop with and hrad51 formation required the and the pBluescript and of the pBluescript DNA with the φX174 DNA d-loop formation has been from biochemical and that Rad54 on of the protein and D. Wyman C. Paulusma C. Kanaar R. Proc. Natl. Acad. Sci. U. S. A. 2001; 84: 8454-8460Crossref Scopus (110) Google Scholar, Komen S. Petukhova G. Sigurdsson S. Stratton S. Sung P. Mol. Cell. 2000; 6: 563-572Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). a of interaction with the ATPase, DNA supercoiling, and DNA strand opening activities of Rad54 are greatly Petukhova (19Petukhova G. Stratton S. Sung P. Nature. 1998; 393: 91-94Crossref PubMed Scopus (345) Google first that homologous DNA pairing by Here we have biochemical that hRad51 and hRad54 work in to make DNA the reaction a of joint formation and We have that ATP hydrolysis by hRad51 have in its from the bound This have to the of to the strand in the d-loop to a of homologous pairing with the DNA The pairing reaction have for the of the with this the ATP hrad51 K133R mutant is at the studies in yeast and cells with the same Rad51 ATPase mutant have shown that is that an level of this mutant is for of the various of cells C. A. E. Y. M. S. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, P. Stratton S.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The that the hrad51 K133R mutant is the protein in reaction that the C. A. E. Y. M. S. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, P. Stratton S.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google and P. A.J. Moynahan M.E. N. Jasin M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of this protein are of a a ability to homologous The hrad51 K133R mutant form a complex with the of protein for recombination reactions. Importantly, our biochemical results that members of the function to of the d-loop reaction by of the hRad54 ATPase the ability to recombination in T.L. Essers J. Citterio E. Swagemakers S.M. de Wit J. Benson F.E. Hoeijmakers J.H. Kanaar R. Curr. Biol. 1999; 9: 325-328Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), with the that the ATP DNA supercoiling and DNA strand opening activities of hRad54 are for recombination. and D. Wyman C. Paulusma C. Kanaar R. Proc. Natl. Acad. Sci. U. S. A. 2001; 84: 8454-8460Crossref Scopus (110) Google Scholar, Komen S. Petukhova G. Sigurdsson S. Stratton S. Sung P. Mol. Cell. 2000; 6: 563-572Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, J.A. S.C. Mol. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar), is that the DNA strand opening of hRad54 the of an DNA for the formation of the DNA joint that The ability of hRad54 to the duplex molecule through its is to the at which the duplex by the filament for a that the DNA by the of hRad51 and hRad54 are critical for the of during recombination. We for with plasmid