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Holliday Junction Binding Activity of the Human Rad51B Protein

2003; Elsevier BV; Volume: 278; Issue: 4 Linguagem: Inglês

10.1074/jbc.m210899200

ISSN

1083-351X

Autores

Hiroshi Yokoyama, Hitoshi Kurumizaka, S Ikawa, Shigeyuki Yokoyama, Takehiko Shibata,

Tópico(s)

Genomics and Chromatin Dynamics

Resumo

The human Rad51B protein is involved in the recombinational repair of damaged DNA. Chromosomal rearrangements of the Rad51B gene have been found in uterine leiomyoma patients, suggesting that the Rad51B gene suppresses tumorigenesis. In the present study, we found that the purified Rad51B protein bound to single-stranded DNA and double-stranded DNA in the presence of ATP and either Mg2+or Mn2+ and hydrolyzed ATP in a DNA-dependent manner. When the synthetic Holliday junction was present along with the half-cruciform and double-stranded oligonucleotides, the Rad51B protein only bound to the synthetic Holliday junction, which mimics a key intermediate in homologous recombination. In contrast, the human Rad51 protein bound to all three DNA substrates with no obvious preference. Therefore, the Rad51B protein may have a specific function in Holliday junction processing in the homologous recombinational repair pathway in humans. The human Rad51B protein is involved in the recombinational repair of damaged DNA. Chromosomal rearrangements of the Rad51B gene have been found in uterine leiomyoma patients, suggesting that the Rad51B gene suppresses tumorigenesis. In the present study, we found that the purified Rad51B protein bound to single-stranded DNA and double-stranded DNA in the presence of ATP and either Mg2+or Mn2+ and hydrolyzed ATP in a DNA-dependent manner. When the synthetic Holliday junction was present along with the half-cruciform and double-stranded oligonucleotides, the Rad51B protein only bound to the synthetic Holliday junction, which mimics a key intermediate in homologous recombination. In contrast, the human Rad51 protein bound to all three DNA substrates with no obvious preference. Therefore, the Rad51B protein may have a specific function in Holliday junction processing in the homologous recombinational repair pathway in humans. homologous recombinational repair single-stranded double-stranded bovine serum albumin high pressure liquid chromatography Tris-acetate/EDTA Tris-borate/EDTA sodium lauryl sulfate Chromosomes are continuously subjected to attacks by exogenous and endogenous mutagens, which damage the genomic DNA. Chromosomal double strand breaks, which are potential inducers of chromosome aberrations and tumorigenesis, are caused by ionizing radiation, oxygen free-radicals, DNA cross-linking reagents, and DNA replication failure (1Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (861) Google Scholar, 2Pierce A.J. Stark J.M. Araujo F.D. Moynahan M.E. Berwick M. Jasin M. Trends Cell Biol. 2001; 11: S52-S58Abstract Full Text PDF PubMed Scopus (235) Google Scholar). Homologous recombinational repair (HRR)1 is an accurate pathway for repair without base substitutions, deletions, and insertions, and therefore, it is important to maintain chromosomal integrity (3Thompson L.H. Schild D. Mutat. Res. 2001; 477: 131-153Crossref PubMed Scopus (362) Google Scholar, 4Ferguson D.O. Alt F.W. Oncogene. 2001; 20: 5572-5579Crossref PubMed Scopus (273) Google Scholar, 5van Gent D.C. Hoeijmarkers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (951) Google Scholar). In the HRR pathway, a single-stranded DNA (ssDNA) tail, which is produced at the site, invades a homologous region of the intact sister chromatid. Through this “homologous-pairing” step, an intermediate structure, the Holliday junction, in which two double-stranded DNA (dsDNA) molecules form a four-way junction (6Holliday R. Genet. Res. 1964; 5: 282-304Crossref Scopus (1261) Google Scholar), is generated between the damaged and intact chromatids (7Lusetti S.L. Cox M.M. Annu. Rev. Biochem. 2002; 71: 71-100Crossref PubMed Scopus (349) Google Scholar). The Holliday junction can move along the DNA and has the ability to expand a heteroduplex region. This step, termed “branch migration,” is essential for the HRR pathway.In humans, genes involved in the HRR pathway, includingRad51, Rad52, Rad54,Rad54B, Brca1, Brca2,Xrcc2, Xrcc3,Rad51B/Rad51L1/hRec2(Rad51B), Rad51C/Rad51L2(Rad51C), and Rad51D/Rad51L3(Rad51D), have been identified (8Thacker J. Biochimie (Paris). 1999; 81: 77-85Crossref PubMed Scopus (62) Google Scholar, 9Wood R.D. Mitchell M. Sgouros J. Lindahl T. Science. 2001; 291: 1284-1289Crossref PubMed Scopus (1098) Google Scholar). Mutations in several of these genes have been found in cancer patients but not in healthy individuals (2Pierce A.J. Stark J.M. Araujo F.D. Moynahan M.E. Berwick M. Jasin M. Trends Cell Biol. 2001; 11: S52-S58Abstract Full Text PDF PubMed Scopus (235) Google Scholar). Notably, gross chromosomal rearrangements of theRad51B gene have been reported in uterine leiomyomas, involving translocations with the HMG1C gene (10Schoenmakers E.F.P.M. Huysmans C. van de Ven W.J.M. Cancer Res. 1999; 59: 19-23PubMed Google Scholar, 11Takahashi T. Nagai N. Oda H. Ohama K. Kamada N. Miyagawa K. Genes Chromosomes Cancer. 2001; 30: 196-201Crossref PubMed Scopus (39) Google Scholar), and in a pulmonary chondroid hamartoma (12Blank C. Schoenmakers E.F.P.M. Rogalla P. Huys E.H.L.P.G. van Rijk A.A.F. Drieschner N. Bullerdiek J. Cytogenet. Cell Genet. 2001; 95: 17-19Crossref PubMed Scopus (12) Google Scholar). In mice, disruption of theRad51B gene results in early embryonic lethality (13Shu Z. Smith S. Wang L. Rice M.C. Kmiec E.B. Mol. Cell. Biol. 1999; 19: 8686-8693Crossref PubMed Scopus (132) Google Scholar), suggesting that the Rad51B gene product is essential for development. The Rad51B knockout in chicken DT40 cells impaired the HRR pathway and caused sensitivity to DNA-damaging agents such as cisplatin, mitomycin C, and γ-rays (14Takata M. Sasaki M.S. Sonoda E. Fukushima T. Morrison C. Albala J.S. Swagemakers S.M. Kanaar R. Thompson L.H. Takeda S. Mol. Cell. Biol. 2000; 20: 6476-6482Crossref PubMed Scopus (224) Google Scholar). Therefore, theRad51B gene product plays an essential role in the HRR pathway in vertebrates. The human Rad51B protein is composed of 350 amino acid residues (15Albala J.S. Thelen M.P. Prange C. Fan W. Christensen M. Thompson L.H. Lennon G.G. Genomics. 1997; 46: 476-479Crossref PubMed Scopus (104) Google Scholar, 16Cartwright R. Dunn A.M. Simpson P.J. Tambini C.E. Thacker J. Nucleic Acids Res. 1998; 26: 1653-1659Crossref PubMed Scopus (74) Google Scholar) and has about 20% amino acid identity with the human Rad51 protein, like other human proteins such as the Xrcc2, Xrcc3, Rad51C, and Rad51D proteins (17Thaker J. Trends Genet. 1999; 15: 166-168Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Therefore, these five proteins, Xrcc2, Xrcc3, Rad51B, Rad51C, and Rad51D, which have similarity with Rad51, have been classified as Rad51 paralogs.In the present study, we purified the human Rad51B protein as a recombinant protein and biochemically characterized it. The purified Rad51B protein bound to ssDNA and dsDNA in the presence of ATP and Mg2+ or Mn2+ and hydrolyzed ATP in a DNA-dependent manner. When the Holliday structure coexisted with the replication-fork-like structure and B-form DNA, Rad51B only bound to the synthetic Holliday junction, suggesting that Rad51B may play a role in Holliday junction processing in the HRR pathway.DISCUSSIONAs bacterial Holliday junction-binding proteins, the RuvA·RuvB complex (RuvAB), which promotes branch migration of the Holliday junction, has been found (19Iwasaki H. Takahagi M. Nakata A. Shinagawa H. Genes Dev. 1992; 6: 2214-2220Crossref PubMed Scopus (148) Google Scholar, 40Shiba T. Iwasaki H. Nakata A. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8445-8449Crossref PubMed Scopus (94) Google Scholar, 41Tsaneva I.R. Muller B. West S.C. Cell. 1992; 69: 1171-1180Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 42Parsons C.A. Tsaneva I. Lloyd R.G. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5452-5456Crossref PubMed Scopus (152) Google Scholar). Crystallographic and electron micrographic analyses have shown that RuvA forms a tetramer, whereas RuvB forms hexameric and heptameric ring structures (43Stasiak A. Tsaneva I.R. West S.C. Benson C.J.B., Yu, X. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7618-7622Crossref PubMed Scopus (143) Google Scholar, 44Parsons C.A. Stasiak A. Bennett R.J. West S.C. Nature. 1995; 374: 375-378Crossref PubMed Scopus (153) Google Scholar, 45Rafferty J.B. Sedelnikova S.E. Hargreaves D. Artymiuk P.J. Baker P.J. Sharples G.J. Mahdi A.A. Lloyd R.G. Rice D.W. Science. 1996; 274: 415-421Crossref PubMed Scopus (150) Google Scholar, 46Yu X. West S.C. Egelman E.H. J. Mol. Biol. 1997; 266: 217-222Crossref PubMed Scopus (67) Google Scholar, 47Hargreaves D. Rice D.W. Sedelnikova S.E. Artymiuk P.J. Lloyd R.G. Rafferty J.B. Nat. Struct. Biol. 1998; 5: 441-446Crossref PubMed Scopus (129) Google Scholar, 48Ariyoshi M. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8257-8262Crossref PubMed Scopus (119) Google Scholar, 49Miyata T. Yamada K. Iwasaki H. Shinagawa H. Morikawa K. Mayanagi K. J. Struct. Biol. 2000; 131: 83-89Crossref PubMed Scopus (48) Google Scholar, 50Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar, 51Putnam C.D. Clancy S.B. Tsuruta H. Gonzalez S. Wetmur J.G. Tainer J.A. J. Mol. Biol. 2001; 311: 297-310Crossref PubMed Scopus (145) Google Scholar, 52Chen Y.-J., Yu, X. Egelman E.H. J. Mol. Biol. 2002; 319: 587-591Crossref PubMed Scopus (22) Google Scholar, 53Yamada K. Miyata T. Tsuchiya D. Oyama T. Fujiwara Y. Ohnishi T. Iwasaki H. Shinagawa H. Ariyoshi M. Mayanagi K. Morikawa K. Mol. Cell. 2002; 10: 671-681Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Both RuvA and RuvB are ATP-binding proteins, and the branch migration promoted by the RuvAB complex requires ATP (19Iwasaki H. Takahagi M. Nakata A. Shinagawa H. Genes Dev. 1992; 6: 2214-2220Crossref PubMed Scopus (148) Google Scholar, 40Shiba T. Iwasaki H. Nakata A. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8445-8449Crossref PubMed Scopus (94) Google Scholar, 41Tsaneva I.R. Muller B. West S.C. Cell. 1992; 69: 1171-1180Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 54Tsaneva I.R. Muller B. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1315-1319Crossref PubMed Scopus (136) Google Scholar). In addition, theE. coli RecG protein is a factor that creates a four-way junction by reversing the stalled replication fork (55McGlynn P. Lloyd R.G. Cell. 2000; 101: 35-45Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 56Singleton M.R. Scaife S. Wigley D.B. Cell. 2001; 107: 79-89Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). In the present study we found that Rad51B specifically binds to the synthetic Holliday junction, suggesting that Rad51B may have a role in processing the Holliday-junction intermediate in HRR. However, we could not detect branch migration activity with Rad51B (data not shown). Helicase activity, which is required to promote efficient branch migration by the RuvAB complex or RecG, also was not detected with Rad51B (data not shown). Rad51B may require a subunit(s), like the bacterial RuvAB complex, if it is involved in the branch migration process in the HRR pathway.Thus far, a eukaryotic nuclease, Mus81, which resolves the Holliday junction into two duplex DNAs, has been suggested to be a functional homolog of the bacterial Holliday-junction resolvase, the RuvC protein (57Boddy M.N. Gaillard P.-H.L. McDonald W.H. Shanahan P Yates III, J.R. Russell P. Cell. 2001; 107: 537-548Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 58Chen X.B. Melchionna R. Denis C.-M. Gaillard P.-H.L. Blasina A. van de Weyer I. Boddy M.N. Russel P. Vialard J. McGowan C.H. Mol. Cell. 2001; 8: 1117-1127Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). On the other hand, an ATP-dependent branch migration activity, which co-fractionates with the Holliday junction resolving activity, has been detected in mammalian cell-free extracts (59Constantinou A. Davies A.A. West S.C. Cell. 2001; 104: 259-268Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 60Constantinou A. Chen X.-B. McGowan C.H. West S.C. EMBO J. 2002; 21: 5577-5585Crossref PubMed Scopus (134) Google Scholar), suggesting that the Holliday junction migrase and resolvase may form a complex in mammals, like the bacterial RuvA, RuvB, and RuvC proteins (61van Gool A.J. Hajibagheri N.M. Stasiak A. West S.C. Genes Dev. 1999; 13: 1861-1870Crossref PubMed Scopus (77) Google Scholar). Rad51B, which can target the Holliday junction structure, may be a subunit of the human Holliday junction-processing complex. In humans, the TIP60 complex, which is composed of fourteen subunits, was found to bind the Holliday junction structure and exhibited helicase activity, but not branch-migration activity (62Ikura T. Ogryzko V.V. Grigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar). The TIP60 complex contains TIP49a (also known as RUVBL1, NMP238, or Pontin52) and TIP49b, which have similarity to RuvB (63Qiu X.B. Lin Y.L. Thome K.C. Pian P. Schlegel B.P. Weremowicz S. Parvin J.D. Dutta A. J. Biol. Chem. 1998; 273: 27786-27793Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 64Holzmann K. Gerner C. Korosec T. Poltl A. Grimm R. Sauermann G. Biochem. Biophys. Res. Commun. 1998; 252: 39-45Crossref PubMed Scopus (39) Google Scholar, 65Bauer A. Huber O. Kemler R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14787-14792Crossref PubMed Scopus (168) Google Scholar, 66Makino Y. Kanemaki M. Kurokawa Y. Koji T. Tamura T. J. Biol. Chem. 1999; 274: 15329-15335Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 67Kanemaki M. Kurokawa Y. Matsu-ura T. Makino Y. Masani A. Okazaki K. Morishita T. Tamura T. J. Biol. Chem. 1999; 274: 22437-22444Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Rad51B may function in the Holliday junction processing with the TIP60 complex. Further studies are required to identify the factors that process the Holliday junction during HRR in mammals. Chromosomes are continuously subjected to attacks by exogenous and endogenous mutagens, which damage the genomic DNA. Chromosomal double strand breaks, which are potential inducers of chromosome aberrations and tumorigenesis, are caused by ionizing radiation, oxygen free-radicals, DNA cross-linking reagents, and DNA replication failure (1Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (861) Google Scholar, 2Pierce A.J. Stark J.M. Araujo F.D. Moynahan M.E. Berwick M. Jasin M. Trends Cell Biol. 2001; 11: S52-S58Abstract Full Text PDF PubMed Scopus (235) Google Scholar). Homologous recombinational repair (HRR)1 is an accurate pathway for repair without base substitutions, deletions, and insertions, and therefore, it is important to maintain chromosomal integrity (3Thompson L.H. Schild D. Mutat. Res. 2001; 477: 131-153Crossref PubMed Scopus (362) Google Scholar, 4Ferguson D.O. Alt F.W. Oncogene. 2001; 20: 5572-5579Crossref PubMed Scopus (273) Google Scholar, 5van Gent D.C. Hoeijmarkers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (951) Google Scholar). In the HRR pathway, a single-stranded DNA (ssDNA) tail, which is produced at the site, invades a homologous region of the intact sister chromatid. Through this “homologous-pairing” step, an intermediate structure, the Holliday junction, in which two double-stranded DNA (dsDNA) molecules form a four-way junction (6Holliday R. Genet. Res. 1964; 5: 282-304Crossref Scopus (1261) Google Scholar), is generated between the damaged and intact chromatids (7Lusetti S.L. Cox M.M. Annu. Rev. Biochem. 2002; 71: 71-100Crossref PubMed Scopus (349) Google Scholar). The Holliday junction can move along the DNA and has the ability to expand a heteroduplex region. This step, termed “branch migration,” is essential for the HRR pathway. In humans, genes involved in the HRR pathway, includingRad51, Rad52, Rad54,Rad54B, Brca1, Brca2,Xrcc2, Xrcc3,Rad51B/Rad51L1/hRec2(Rad51B), Rad51C/Rad51L2(Rad51C), and Rad51D/Rad51L3(Rad51D), have been identified (8Thacker J. Biochimie (Paris). 1999; 81: 77-85Crossref PubMed Scopus (62) Google Scholar, 9Wood R.D. Mitchell M. Sgouros J. Lindahl T. Science. 2001; 291: 1284-1289Crossref PubMed Scopus (1098) Google Scholar). Mutations in several of these genes have been found in cancer patients but not in healthy individuals (2Pierce A.J. Stark J.M. Araujo F.D. Moynahan M.E. Berwick M. Jasin M. Trends Cell Biol. 2001; 11: S52-S58Abstract Full Text PDF PubMed Scopus (235) Google Scholar). Notably, gross chromosomal rearrangements of theRad51B gene have been reported in uterine leiomyomas, involving translocations with the HMG1C gene (10Schoenmakers E.F.P.M. Huysmans C. van de Ven W.J.M. Cancer Res. 1999; 59: 19-23PubMed Google Scholar, 11Takahashi T. Nagai N. Oda H. Ohama K. Kamada N. Miyagawa K. Genes Chromosomes Cancer. 2001; 30: 196-201Crossref PubMed Scopus (39) Google Scholar), and in a pulmonary chondroid hamartoma (12Blank C. Schoenmakers E.F.P.M. Rogalla P. Huys E.H.L.P.G. van Rijk A.A.F. Drieschner N. Bullerdiek J. Cytogenet. Cell Genet. 2001; 95: 17-19Crossref PubMed Scopus (12) Google Scholar). In mice, disruption of theRad51B gene results in early embryonic lethality (13Shu Z. Smith S. Wang L. Rice M.C. Kmiec E.B. Mol. Cell. Biol. 1999; 19: 8686-8693Crossref PubMed Scopus (132) Google Scholar), suggesting that the Rad51B gene product is essential for development. The Rad51B knockout in chicken DT40 cells impaired the HRR pathway and caused sensitivity to DNA-damaging agents such as cisplatin, mitomycin C, and γ-rays (14Takata M. Sasaki M.S. Sonoda E. Fukushima T. Morrison C. Albala J.S. Swagemakers S.M. Kanaar R. Thompson L.H. Takeda S. Mol. Cell. Biol. 2000; 20: 6476-6482Crossref PubMed Scopus (224) Google Scholar). Therefore, theRad51B gene product plays an essential role in the HRR pathway in vertebrates. The human Rad51B protein is composed of 350 amino acid residues (15Albala J.S. Thelen M.P. Prange C. Fan W. Christensen M. Thompson L.H. Lennon G.G. Genomics. 1997; 46: 476-479Crossref PubMed Scopus (104) Google Scholar, 16Cartwright R. Dunn A.M. Simpson P.J. Tambini C.E. Thacker J. Nucleic Acids Res. 1998; 26: 1653-1659Crossref PubMed Scopus (74) Google Scholar) and has about 20% amino acid identity with the human Rad51 protein, like other human proteins such as the Xrcc2, Xrcc3, Rad51C, and Rad51D proteins (17Thaker J. Trends Genet. 1999; 15: 166-168Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Therefore, these five proteins, Xrcc2, Xrcc3, Rad51B, Rad51C, and Rad51D, which have similarity with Rad51, have been classified as Rad51 paralogs. In the present study, we purified the human Rad51B protein as a recombinant protein and biochemically characterized it. The purified Rad51B protein bound to ssDNA and dsDNA in the presence of ATP and Mg2+ or Mn2+ and hydrolyzed ATP in a DNA-dependent manner. When the Holliday structure coexisted with the replication-fork-like structure and B-form DNA, Rad51B only bound to the synthetic Holliday junction, suggesting that Rad51B may play a role in Holliday junction processing in the HRR pathway. DISCUSSIONAs bacterial Holliday junction-binding proteins, the RuvA·RuvB complex (RuvAB), which promotes branch migration of the Holliday junction, has been found (19Iwasaki H. Takahagi M. Nakata A. Shinagawa H. Genes Dev. 1992; 6: 2214-2220Crossref PubMed Scopus (148) Google Scholar, 40Shiba T. Iwasaki H. Nakata A. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8445-8449Crossref PubMed Scopus (94) Google Scholar, 41Tsaneva I.R. Muller B. West S.C. Cell. 1992; 69: 1171-1180Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 42Parsons C.A. Tsaneva I. Lloyd R.G. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5452-5456Crossref PubMed Scopus (152) Google Scholar). Crystallographic and electron micrographic analyses have shown that RuvA forms a tetramer, whereas RuvB forms hexameric and heptameric ring structures (43Stasiak A. Tsaneva I.R. West S.C. Benson C.J.B., Yu, X. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7618-7622Crossref PubMed Scopus (143) Google Scholar, 44Parsons C.A. Stasiak A. Bennett R.J. West S.C. Nature. 1995; 374: 375-378Crossref PubMed Scopus (153) Google Scholar, 45Rafferty J.B. Sedelnikova S.E. Hargreaves D. Artymiuk P.J. Baker P.J. Sharples G.J. Mahdi A.A. Lloyd R.G. Rice D.W. Science. 1996; 274: 415-421Crossref PubMed Scopus (150) Google Scholar, 46Yu X. West S.C. Egelman E.H. J. Mol. Biol. 1997; 266: 217-222Crossref PubMed Scopus (67) Google Scholar, 47Hargreaves D. Rice D.W. Sedelnikova S.E. Artymiuk P.J. Lloyd R.G. Rafferty J.B. Nat. Struct. Biol. 1998; 5: 441-446Crossref PubMed Scopus (129) Google Scholar, 48Ariyoshi M. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8257-8262Crossref PubMed Scopus (119) Google Scholar, 49Miyata T. Yamada K. Iwasaki H. Shinagawa H. Morikawa K. Mayanagi K. J. Struct. Biol. 2000; 131: 83-89Crossref PubMed Scopus (48) Google Scholar, 50Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar, 51Putnam C.D. Clancy S.B. Tsuruta H. Gonzalez S. Wetmur J.G. Tainer J.A. J. Mol. Biol. 2001; 311: 297-310Crossref PubMed Scopus (145) Google Scholar, 52Chen Y.-J., Yu, X. Egelman E.H. J. Mol. Biol. 2002; 319: 587-591Crossref PubMed Scopus (22) Google Scholar, 53Yamada K. Miyata T. Tsuchiya D. Oyama T. Fujiwara Y. Ohnishi T. Iwasaki H. Shinagawa H. Ariyoshi M. Mayanagi K. Morikawa K. Mol. Cell. 2002; 10: 671-681Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Both RuvA and RuvB are ATP-binding proteins, and the branch migration promoted by the RuvAB complex requires ATP (19Iwasaki H. Takahagi M. Nakata A. Shinagawa H. Genes Dev. 1992; 6: 2214-2220Crossref PubMed Scopus (148) Google Scholar, 40Shiba T. Iwasaki H. Nakata A. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8445-8449Crossref PubMed Scopus (94) Google Scholar, 41Tsaneva I.R. Muller B. West S.C. Cell. 1992; 69: 1171-1180Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 54Tsaneva I.R. Muller B. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1315-1319Crossref PubMed Scopus (136) Google Scholar). In addition, theE. coli RecG protein is a factor that creates a four-way junction by reversing the stalled replication fork (55McGlynn P. Lloyd R.G. Cell. 2000; 101: 35-45Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 56Singleton M.R. Scaife S. Wigley D.B. Cell. 2001; 107: 79-89Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). In the present study we found that Rad51B specifically binds to the synthetic Holliday junction, suggesting that Rad51B may have a role in processing the Holliday-junction intermediate in HRR. However, we could not detect branch migration activity with Rad51B (data not shown). Helicase activity, which is required to promote efficient branch migration by the RuvAB complex or RecG, also was not detected with Rad51B (data not shown). Rad51B may require a subunit(s), like the bacterial RuvAB complex, if it is involved in the branch migration process in the HRR pathway.Thus far, a eukaryotic nuclease, Mus81, which resolves the Holliday junction into two duplex DNAs, has been suggested to be a functional homolog of the bacterial Holliday-junction resolvase, the RuvC protein (57Boddy M.N. Gaillard P.-H.L. McDonald W.H. Shanahan P Yates III, J.R. Russell P. Cell. 2001; 107: 537-548Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 58Chen X.B. Melchionna R. Denis C.-M. Gaillard P.-H.L. Blasina A. van de Weyer I. Boddy M.N. Russel P. Vialard J. McGowan C.H. Mol. Cell. 2001; 8: 1117-1127Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). On the other hand, an ATP-dependent branch migration activity, which co-fractionates with the Holliday junction resolving activity, has been detected in mammalian cell-free extracts (59Constantinou A. Davies A.A. West S.C. Cell. 2001; 104: 259-268Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 60Constantinou A. Chen X.-B. McGowan C.H. West S.C. EMBO J. 2002; 21: 5577-5585Crossref PubMed Scopus (134) Google Scholar), suggesting that the Holliday junction migrase and resolvase may form a complex in mammals, like the bacterial RuvA, RuvB, and RuvC proteins (61van Gool A.J. Hajibagheri N.M. Stasiak A. West S.C. Genes Dev. 1999; 13: 1861-1870Crossref PubMed Scopus (77) Google Scholar). Rad51B, which can target the Holliday junction structure, may be a subunit of the human Holliday junction-processing complex. In humans, the TIP60 complex, which is composed of fourteen subunits, was found to bind the Holliday junction structure and exhibited helicase activity, but not branch-migration activity (62Ikura T. Ogryzko V.V. Grigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar). The TIP60 complex contains TIP49a (also known as RUVBL1, NMP238, or Pontin52) and TIP49b, which have similarity to RuvB (63Qiu X.B. Lin Y.L. Thome K.C. Pian P. Schlegel B.P. Weremowicz S. Parvin J.D. Dutta A. J. Biol. Chem. 1998; 273: 27786-27793Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 64Holzmann K. Gerner C. Korosec T. Poltl A. Grimm R. Sauermann G. Biochem. Biophys. Res. Commun. 1998; 252: 39-45Crossref PubMed Scopus (39) Google Scholar, 65Bauer A. Huber O. Kemler R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14787-14792Crossref PubMed Scopus (168) Google Scholar, 66Makino Y. Kanemaki M. Kurokawa Y. Koji T. Tamura T. J. Biol. Chem. 1999; 274: 15329-15335Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 67Kanemaki M. Kurokawa Y. Matsu-ura T. Makino Y. Masani A. Okazaki K. Morishita T. Tamura T. J. Biol. Chem. 1999; 274: 22437-22444Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). 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Both RuvA and RuvB are ATP-binding proteins, and the branch migration promoted by the RuvAB complex requires ATP (19Iwasaki H. Takahagi M. Nakata A. Shinagawa H. Genes Dev. 1992; 6: 2214-2220Crossref PubMed Scopus (148) Google Scholar, 40Shiba T. Iwasaki H. Nakata A. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8445-8449Crossref PubMed Scopus (94) Google Scholar, 41Tsaneva I.R. Muller B. West S.C. Cell. 1992; 69: 1171-1180Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 54Tsaneva I.R. Muller B. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1315-1319Crossref PubMed Scopus (136) Google Scholar). In addition, theE. coli RecG protein is a factor that creates a four-way junction by reversing the stalled replication fork (55McGlynn P. Lloyd R.G. Cell. 2000; 101: 35-45Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 56Singleton M.R. Scaife S. Wigley D.B. Cell. 2001; 107: 79-89Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). In the present study we found that Rad51B specifically binds to the synthetic Holliday junction, suggesting that Rad51B may have a role in processing the Holliday-junction intermediate in HRR. However, we could not detect branch migration activity with Rad51B (data not shown). Helicase activity, which is required to promote efficient branch migration by the RuvAB complex or RecG, also was not detected with Rad51B (data not shown). Rad51B may require a subunit(s), like the bacterial RuvAB complex, if it is involved in the branch migration process in the HRR pathway. Thus far, a eukaryotic nuclease, Mus81, which resolves the Holliday junction into two duplex DNAs, has been suggested to be a functional homolog of the bacterial Holliday-junction resolvase, the RuvC protein (57Boddy M.N. Gaillard P.-H.L. McDonald W.H. Shanahan P Yates III, J.R. Russell P. Cell. 2001; 107: 537-548Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 58Chen X.B. Melchionna R. Denis C.-M. Gaillard P.-H.L. Blasina A. van de Weyer I. Boddy M.N. Russel P. Vialard J. McGowan C.H. Mol. Cell. 2001; 8: 1117-1127Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). On the other hand, an ATP-dependent branch migration activity, which co-fractionates with the Holliday junction resolving activity, has been detected in mammalian cell-free extracts (59Constantinou A. Davies A.A. West S.C. Cell. 2001; 104: 259-268Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 60Constantinou A. Chen X.-B. McGowan C.H. West S.C. EMBO J. 2002; 21: 5577-5585Crossref PubMed Scopus (134) Google Scholar), suggesting that the Holliday junction migrase and resolvase may form a complex in mammals, like the bacterial RuvA, RuvB, and RuvC proteins (61van Gool A.J. Hajibagheri N.M. Stasiak A. West S.C. Genes Dev. 1999; 13: 1861-1870Crossref PubMed Scopus (77) Google Scholar). Rad51B, which can target the Holliday junction structure, may be a subunit of the human Holliday junction-processing complex. In humans, the TIP60 complex, which is composed of fourteen subunits, was found to bind the Holliday junction structure and exhibited helicase activity, but not branch-migration activity (62Ikura T. Ogryzko V.V. Grigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar). The TIP60 complex contains TIP49a (also known as RUVBL1, NMP238, or Pontin52) and TIP49b, which have similarity to RuvB (63Qiu X.B. Lin Y.L. Thome K.C. Pian P. Schlegel B.P. Weremowicz S. Parvin J.D. Dutta A. J. Biol. Chem. 1998; 273: 27786-27793Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 64Holzmann K. Gerner C. Korosec T. Poltl A. Grimm R. Sauermann G. Biochem. Biophys. Res. Commun. 1998; 252: 39-45Crossref PubMed Scopus (39) Google Scholar, 65Bauer A. Huber O. Kemler R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14787-14792Crossref PubMed Scopus (168) Google Scholar, 66Makino Y. Kanemaki M. Kurokawa Y. Koji T. Tamura T. J. Biol. Chem. 1999; 274: 15329-15335Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 67Kanemaki M. Kurokawa Y. Matsu-ura T. Makino Y. Masani A. Okazaki K. Morishita T. Tamura T. J. Biol. Chem. 1999; 274: 22437-22444Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Rad51B may function in the Holliday junction processing with the TIP60 complex. Further studies are required to identify the factors that process the Holliday junction during HRR in mammals. We thank Dr. Hiroshi Iwasaki (Yokohama City University) for discussions. We also thank Takashi Kinebuchi, Wataru Kagawa, and Rima Enomoto (RIKEN Genomic Sciences Center) for technical assistance.

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