Functional Interaction between the Bloom's Syndrome Helicase and the RAD51 Paralog, RAD51L3 (RAD51D)
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m308838200
ISSN1083-351X
AutoresJeremy Braybrooke, Jiliang Li, Leonard Wu, Fiona Caple, Fiona E. Benson, Ian D. Hickson,
Tópico(s)Genomics and Chromatin Dynamics
ResumoBloom's syndrome (BS) is a genetic disorder associated with short stature, fertility defects, and a predisposition to the development of cancer. BS cells are characterized by genomic instability; in particular, a high rate of reciprocal exchanges between sister-chromatids and homologous chromosomes. The BS gene product, BLM, is a helicase belonging to the highly conserved RecQ family. BLM is known to form a complex with the RAD51 recombinase, and to act upon DNA intermediates that form during homologous recombination, including D-loops and Holliday junctions. Here, we show that BLM also makes a direct physical association with the RAD51L3 protein (also known as RAD51D), a so-called RAD51 paralog that shows limited sequence similarity to RAD51 itself. This interaction is mediated through the N-terminal domain of BLM. To analyze functional interactions between BLM and RAD51L3, we have purified a heteromeric complex comprising RAD51L3 and a second RAD51 paralog, XRCC2. We show that the RAD51L3-XRCC2 complex stimulates BLM to disrupt synthetic 4-way junctions that model the Holliday junction. We also show that a truncated form of BLM, which retains helicase activity but is unable to bind RAD51L3, is not stimulated by the RAD51L3-XRCC2 complex. Our data indicate that the activity of BLM is modulated through an interaction with the RAD51L3-XRCC2 complex, and that this stimulatory effect on BLM is dependent upon a direct physical association between the BLM and RAD51L3 proteins. We propose that BLM co-operates with RAD51 paralogs during the late stages of homologous recombination processes that serve to restore productive DNA replication at sites of damaged or stalled replication forks. Bloom's syndrome (BS) is a genetic disorder associated with short stature, fertility defects, and a predisposition to the development of cancer. BS cells are characterized by genomic instability; in particular, a high rate of reciprocal exchanges between sister-chromatids and homologous chromosomes. The BS gene product, BLM, is a helicase belonging to the highly conserved RecQ family. BLM is known to form a complex with the RAD51 recombinase, and to act upon DNA intermediates that form during homologous recombination, including D-loops and Holliday junctions. Here, we show that BLM also makes a direct physical association with the RAD51L3 protein (also known as RAD51D), a so-called RAD51 paralog that shows limited sequence similarity to RAD51 itself. This interaction is mediated through the N-terminal domain of BLM. To analyze functional interactions between BLM and RAD51L3, we have purified a heteromeric complex comprising RAD51L3 and a second RAD51 paralog, XRCC2. We show that the RAD51L3-XRCC2 complex stimulates BLM to disrupt synthetic 4-way junctions that model the Holliday junction. We also show that a truncated form of BLM, which retains helicase activity but is unable to bind RAD51L3, is not stimulated by the RAD51L3-XRCC2 complex. Our data indicate that the activity of BLM is modulated through an interaction with the RAD51L3-XRCC2 complex, and that this stimulatory effect on BLM is dependent upon a direct physical association between the BLM and RAD51L3 proteins. We propose that BLM co-operates with RAD51 paralogs during the late stages of homologous recombination processes that serve to restore productive DNA replication at sites of damaged or stalled replication forks. Bloom's syndrome (BS) 1The abbreviations used are: BSBloom's syndromeSCEsister-chromatid exchangesORFopen reading frameDTTdithiothreitolGSTglutathione S-transferasePBSAphosphate-buffered salineBSAbovine serum albuminDSBdouble strand break. is a rare, autosomal recessive disorder characterized by proportional dwarfism, immunodeficiency, subfertility, and a greatly increased incidence of a wide range of cancers (1German J. Dermatol. Clin. 1995; 13: 7-18Abstract Full Text PDF PubMed Google Scholar, 2van Brabant A.J. Stan R. Ellis N.A. Annu. Rev. Genomics Hum. Genet. 2000; 1: 409-459Crossref PubMed Scopus (210) Google Scholar). Cell lines derived from individuals with BS display a number of abnormalities, including poor growth and plating, and a strikingly high level of chromosomal instability (3Karow J.K. Wu L. Hickson I.D. Curr. Opin. Genet. Dev. 2000; 10: 32-38Crossref PubMed Scopus (162) Google Scholar, 4Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (582) Google Scholar). The characteristic feature of BS cells, which is used in diagnosis of the disorder, is an elevated frequency of genetic recombination events, particularly sister-chromatid exchanges (SCEs) (5Chaganti R.S. Schonberg S. German J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4508-4512Crossref PubMed Scopus (793) Google Scholar). However, this hyper-recombination is not limited to exchanges between sister-chromatids, because interchromosomal homologous recombination also occurs at an elevated rate in BS cells (1German J. Dermatol. Clin. 1995; 13: 7-18Abstract Full Text PDF PubMed Google Scholar, 2van Brabant A.J. Stan R. Ellis N.A. Annu. Rev. Genomics Hum. Genet. 2000; 1: 409-459Crossref PubMed Scopus (210) Google Scholar) Bloom's syndrome sister-chromatid exchanges open reading frame dithiothreitol glutathione S-transferase phosphate-buffered saline bovine serum albumin double strand break. The gene mutated in BS, which is designated BLM, is located on chromosome 15q26.1, and encodes a protein comprising 1417 amino acids with a predicted Mr of 159,000 (6Ellis N.A. Groden J. Ye T.Z. Straughen J. Lennon D.J. Ciocci S. Proytcheva M. German J. Cell. 1995; 83: 655-666Abstract Full Text PDF PubMed Scopus (1230) Google Scholar). BLM belongs to the highly conserved RecQ family of DNA helicases (3Karow J.K. Wu L. Hickson I.D. Curr. Opin. Genet. Dev. 2000; 10: 32-38Crossref PubMed Scopus (162) Google Scholar, 4Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (582) Google Scholar). Because members of the RecQ family have been identified in all organisms, it seems likely that they perform an important and conserved cellular function. There is a single family member in bacterial and yeast species, but at least five in human cells. Of the five human RecQ-related proteins, defects in three are associated with established genetic disorders. In addition to inactivation of BLM in BS, defects in WRN cause Werner's syndrome (7Yu C. Oshima J. Fu Y. Wijsman E.M. Hisama F. Alisch R. Matthews S. Najura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1507) Google Scholar), and defects in RECQ4 cause Rothmund-Thomson syndrome (8Kitao S. Shimamoto A. Goto M. Miller R.W. Smithson W.A. Lindor N.M. Furuichi Y. Nat. Genet. 1999; 22: 82-84Crossref PubMed Scopus (585) Google Scholar). Like BS, both of these disorders are associated with a high incidence of cancers, although the primary manifestations of Werner's syndrome and Rothmund-Thomson syndrome are premature aging, and skin and skeletal abnormalities, respectively. Where studied, all RecQ helicases unwind simple, partial-duplex DNA molecules in a 3′-5′ direction but are notable for their atypical profile of preferred DNA substrates (9Mohaghegh P. Karow J.K. Brosh Jr., R.M. Bohr Jr., V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (498) Google Scholar). For example, RecQ helicases, including BLM, are apparently unique among helicases in their ability to efficiently disrupt "alternative" (non-Watson-Crick) DNA structures, including G-quadruplexes, that can form in guanine-rich sequences such as telomeric repeat DNA (9Mohaghegh P. Karow J.K. Brosh Jr., R.M. Bohr Jr., V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (498) Google Scholar, 10Wu X. Maizels N. Nucleic Acids Res. 2001; 29: 1765-1771Crossref PubMed Scopus (95) Google Scholar, 11Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 12Sun H. Bennett R.J. Maizels N. Nucleic Acids Res. 1999; 27: 1978-1984Crossref PubMed Scopus (193) Google Scholar, 13Fry M. Loeb L.A. J. Biol. Chem. 1999; 274: 12797-12802Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Moreover, RecQ helicases selectively bind to at least two DNA structures that arise during the process of homologous recombination. BLM can disrupt synthetic oligonucleotides that mimic the DNA displacement loop (D-loop) structure generated by DNA strand invasion of single-stranded DNA (ssDNA) into an homologous duplex (14van Brabant A.J. Ye T. Sanz M. German I.J. Ellis N.A. Holloman W.K. Biochemistry. 2000; 39: 14617-14625Crossref PubMed Scopus (201) Google Scholar). BLM, WRN, and RecQ also unwind synthetic 4-way junctions (X-junctions) that model the Holliday junction recombination intermediate (9Mohaghegh P. Karow J.K. Brosh Jr., R.M. Bohr Jr., V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (498) Google Scholar, 15Karow J.K. Constantinou A. Li J.L. West S.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6504-6508Crossref PubMed Scopus (424) Google Scholar, 16Harmon F.G. Kowalczykowski S.C. J. Biol. Chem. 2001; 276: 232-243Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), and BLM and WRN have been shown to catalyze efficient and extensive branch migration of RecA-generated Holliday junctions (15Karow J.K. Constantinou A. Li J.L. West S.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6504-6508Crossref PubMed Scopus (424) Google Scholar, 17Constantinou A. Tarsounas M. Karow J.K. Brosh R.M. Bohr V.A. Hickson I.D. West S.C. EMBO Rep. 2000; 1: 80-84Crossref PubMed Scopus (339) Google Scholar). The process of homologous recombination in eukaryotic cells requires several proteins, of which the central player is RAD51, the eukaryotic ortholog of Escherichia coli RecA (18Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar, 19West S.C. Nat. Rev. Mol. Cell. Biol. 2003; 4: 435-445Crossref PubMed Scopus (820) Google Scholar). RAD51 forms helical filaments on DNA and catalyzes DNA strand invasion and exchange. It is assisted in these processes by the actions of several proteins, including RAD52, RAD54, and replication protein A. In Saccharomyces cerevisiae, a heterodimer of the Rad55 and Rad57 proteins acts as a ScRad51 accessory factor, and stimulates ScRad51-mediated DNA strand transfer activity in vitro (20Johnson R.D. Symington L.S. Mol. Cell. Biol. 1995; 15: 4843-4850Crossref PubMed Scopus (212) Google Scholar, 21Sung P. Genes Dev. 1997; 11: 1111-1121Crossref PubMed Scopus (467) Google Scholar). Rad55 and Rad57 are so-called Rad51 paralogs, because they show limited sequence similarity to Rad51, but do not appear to form nucleoprotein filaments or directly catalyze DNA strand invasion/exchange. In human cells, there are five RAD51 paralogs, although it is unclear whether any of these can strictly be considered as the direct counterpart of yeast Rad55 or Rad57. The human RAD51 paralogs are termed RAD51L1 (also known as RAD51B), RAD51L2 (RAD51C), RAD51L3 (RAD51D), XRCC2, and XRCC3 (22Cartwright R. Dunn A.M. Simpson P.J. Tambini C.E. Thacker J. Nucleic Acids Res. 1998; 26: 1653-1659Crossref PubMed Scopus (76) Google Scholar, 23Liu 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, 24Tebbs R.S. Zhao Y. Tucker J.D. Scheerer J.B. Siciliano M.J. Hwang M. Liu N. Legerski R.J. Thompson L.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6354-6358Crossref PubMed Scopus (241) Google Scholar, 25Albala J.S. Thelen M.P. Prange C. Fan W. Christensen M. Thompson L.H. Lennon G.G. Genomics. 1997; 46: 476-479Crossref PubMed Scopus (107) Google Scholar, 26Rice M.C. Smith S.T. Bullrich F. Havre P. Kmiec E.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7417-7422Crossref PubMed Scopus (86) Google Scholar, 27Cartwright R. Tambini C.E. Simpson P.J. Thacker J. Nucleic Acids Res. 1998; 26: 3084-3089Crossref PubMed Scopus (128) Google Scholar, 28Dosanjh M.K. Collins D.W. Fan W. Lennon G.G. Albala J.S. Shen Z. Schild D. Nucleic Acids Res. 1998; 26: 1179-1184Crossref PubMed Scopus (147) Google Scholar, 29Kawabata M. Saeki K. Biochem. Biophys. Res. Commun. 1999; 257: 156-162Crossref PubMed Scopus (20) Google Scholar, 30Pittman D.L. Weinberg L.R. Schimenti J.C. Genomics. 1998; 49: 103-111Crossref PubMed Scopus (114) Google Scholar). The RAD51L2, XRCC2, and XRCC3 proteins are defective in hamster cell mutants (designated irs3, irs1, and irs1SF, respectively) isolated on the basis of hypersensitivity to ionizing radiation (31Thacker J. Zdzienicka M.Z. DNA Repair. 2003; 2: 655-672Crossref PubMed Scopus (108) Google Scholar). These mutants also show hypersensitivity to a variety of other DNA damaging agents, including DNA cross-linking chemicals, as well as extensive genomic instability (23Liu 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, 32Jones N.J. Cox R. Thacker J. Mutat. Res. 1987; 183: 279-286Crossref PubMed Scopus (186) Google Scholar, 33Fuller L.F. Painter R.B. Mutat. Res. 1988; 193: 109-121Crossref PubMed Scopus (166) Google Scholar, 34Griffin C.S. Simpson P.J. Wilson C.R. Thacker J. Nat. Cell Biol. 2000; 2: 757-761Crossref PubMed Scopus (212) Google Scholar). The human RAD51 paralogs form complexes with each other, but not obviously with RAD51 itself. Various complexes have been purified, including RAD51L2-XRCC3, RAD51L3-XRCC2, RAD51L1/L2, and a larger complex containing RAD51L1/L2/L3-XRCC2 (35Kurumizaka 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, 36Masson 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, 37Masson 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, 38Sigurdsson S. Van Komen S. Bussen W. Schild D. Albala J.S. Sung P. Genes Dev. 2001; 15: 3308-3318Crossref PubMed Scopus (190) Google Scholar, 39Liu N. Schild D. Thelen M.P. Thompson L.H. Nucleic Acids Res. 2002; 30: 1009-1015Crossref PubMed Scopus (109) Google Scholar). The biochemical properties of these complexes have not been characterized in detail, and it still remains unclear why five RAD51 paralogs exist in human cells and what their respective functions might be. Apart from an ATPase activity that is stimulated by DNA, little is known about the catalytic activities displayed by the human RAD51 paralogs. One possible role, identified through an analysis of the RAD51L1/L2 complex, is to assist RAD51 in the assembly of a nucleoprotein filament on ssDNA through modulating the action of replication protein A (38Sigurdsson S. Van Komen S. Bussen W. Schild D. Albala J.S. Sung P. Genes Dev. 2001; 15: 3308-3318Crossref PubMed Scopus (190) Google Scholar). Although there is no conclusive evidence for a direct interaction between human RAD51 and the human RAD51 paralogs, it is clear that the action of RAD51 in vivo is influenced by the presence of the paralogs. The most striking manifestation of this is the failure of RAD51 to localize normally to discrete nuclear foci (thought to be sites of ongoing DNA repair) following exposure to ionizing radiation in the irs1, irs1SF, and irs3 cell lines discussed above (40Bishop D.K. Ear U. Bhattacharyya A. Calderone C. Beckett M. Weichselbaum R.R. Shinohara A. J. Biol. Chem. 1998; 273: 21482-21488Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 41French C.A. Masson J.Y. Griffin C.S. O'Regan P. West S.C. Thacker J. J. Biol. Chem. 2002; 277: 19322-19330Abstract Full Text Full Text PDF PubMed Scopus (88) 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). In addition to their promotion of DNA transactions associated with homologous recombination (such as Holliday junction branch migration), RecQ helicases interact physically with components of the homologous recombination machinery. For example, several RecQ helicases associate with and are stimulated by replication protein A (43Brosh Jr., R.M. Orren D.K. Nehlin J.O. Ravn P.H. Kenny M.K. Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 44Brosh Jr., R.M. Li J.L. Kenny M.K. Karow J.K. Cooper M.P. Kureekattil R.P. Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; 275: 23500-23508Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 45Cui S. Klima R. Ochem A. Arosio D. Falaschi A. Vindigni A. J. Biol. Chem. 2003; 278: 1424-1432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). It is not clear, however, whether this is relevant to a role for the complex in DNA recombination or replication. More specifically for recombination, there is a conserved interaction between RecQ helicases and RAD51; both between the human BLM and RAD51 proteins, and between the sole RecQ helicase in S. cerevisiae, Sgs1p, and the ScRad51 protein (46Wu L. Davies S.L. Levitt N.C. Hickson I.D. J. Biol. Chem. 2001; 276: 19375-19381Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). This interaction is mediated by the same domain of the RecQ helicase protein (the extreme C-terminal region in each case) (46Wu L. Davies S.L. Levitt N.C. Hickson I.D. J. Biol. Chem. 2001; 276: 19375-19381Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) despite a lack of obvious sequence similarity between the C-terminal regions of the BLM and Sgs1 proteins. In view of the numerous biochemical and genetic connections between RecQ helicases and components of the homologous recombination machinery, we have analyzed whether BLM might interact with the human RAD51 paralogs. Here, we show that the N-terminal domain of BLM binds to the RAD51L3 protein. To address whether this interaction has functional effects on the catalytic activity of BLM, we have purified RAD51L3 in complex with its RAD51 paralog partner, XRCC2. We show that the RAD51L3-XRCC2 heteromeric complex can stimulate the ability of BLM to disrupt model Holliday junction structures, and that this stimulatory effect requires that BLM be capable of interacting physically with the RAD51L3-XRCC2 complex. We present a model whereby BLM cooperates with the homologous recombination machinery to process DNA structures that arise at sites of blocked replication forks in human cells. Construction of Plasmids—The plasmids pJB1.1, pJB1.2, pJB3.1, and pJB3.2 have been described previously (47Braybrooke J.P. Spink K.G. Thacker J. Hickson I.D. J. Biol. Chem. 2000; 275: 29100-29106Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). These plasmids, respectively, contain the full-length XRCC2 open reading frame (ORF) cloned into pGEX4T-1 and pET30a, or the full-length RAD51L3 ORF cloned into pET30a and pGEX4T-1. pJB3.21 was generated by cloning the full-length open reading frame for RAD51L3 into pET21a using EcoRI and NotI sites. The 3′ primer contained an in-frame stop codon. For two-hybrid analyses, previously reported 3′ constructs of the BLM cDNA were utilized (48Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9636-9644Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). These encode the C-terminal domain of BLM from residues 966 to 1417. The RAD51L3 and XRCC2 ORFs were cloned directionally (using 5′ EcoRI and 3′ NotI sites) into the pEG202 yeast two-hybrid "bait" plasmid, to generate pEG3.1 and pEG1.1, respectively. The same ORFs were also cloned into the EcoRI site of the 2-hybrid "prey" plasmid, pJG45, to generate pJG3.1 and pJG1.1, respectively. The oligonucleotides used in the PCR amplification of the ORFs can be obtained upon request. All constructs were verified by DNA sequencing. Yeast Two-hybrid Analysis—Yeast two-hybrid screens were conducted essentially as described by Wu et al. (46Wu L. Davies S.L. Levitt N.C. Hickson I.D. J. Biol. Chem. 2001; 276: 19375-19381Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Briefly, the XRCC2 or RAD51L3 cDNAs were cloned into the plasmid pEG202 (a gift from Dr. R. Brent) to allow expression of XRCC2 or RAD51L3 bait protein as a fusion to LexA under control of the ADH promoter. RAD51L3 or XRCC2 were also cloned into plasmid pJG45 (a gift from Dr. R. Brent) to create a translational fusion prey under control of the GAL1 galactose inducible promoter (49Brent R. Ptashne M. Cell. 1985; 43: 729-736Abstract Full Text PDF PubMed Scopus (457) Google Scholar). Plasmids were transformed into yeast strain EGY48, containing the reporter plasmid pSH1834, using the lithium acetate method, as published previously (50Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2930) Google Scholar). Transformants were selected at 30 °C on SD-agar lacking histidine, tryptophan, and uracil. Colonies were streaked onto YPD agar plates, which were incubated at 30 °C overnight, before colonies were replica plated onto SD-agar containing galactose, raffinose, and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal), but lacking histidine, tryptophan, uracil, and leucine. Colonies were considered positive if they grew and turned blue, after overnight incubation at 30 °C. Control proteins, including hRAD51, LexMax, and HM12, were used as preys to assess the specificity of the interaction (51Zervos A.S. Gyuris J. Brent R. Cell. 1993; 72: 223-232Abstract Full Text PDF PubMed Scopus (672) Google Scholar). Further two-hybrid experiments were performed, using fragments of BLM (see Fig. 1) as either bait or prey proteins. Quantitative β-galactosidase assays on liquid cultures were performed as described previously (47Braybrooke J.P. Spink K.G. Thacker J. Hickson I.D. J. Biol. Chem. 2000; 275: 29100-29106Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Preparation of Extracts from Human Cell Lines—Whole cell extracts from human cells were prepared by washing cells in PBSA and then boiling in SDS-PAGE protein loading buffer. Nuclear extracts were prepared from exponentially growing HeLa S3 cells in suspension. Approximately 2 × 108 cells were harvested by centrifugation, the pellet was washed in PBSA, and the cells were lysed in 5 ml of buffer (10 mm Tris-HCl, pH 7.5, 1.5 mm MgCl2, 10 mm NaCl, 1% (v/v) Nonidet P-40, 1 mm DTT), supplemented with protease and phosphatase inhibitors (1 mm sodium fluoride, 1 mm β-glycerophosphate, 1 mm sodium orthovanadate, 5 mm sodium pyrophosphate, 1 mm glucose 1-phosphate, 10 nm microcystin, 0.1 mmpara-nitrophenylphosphate, 1 mm phenylmethylsulfonyl fluoride, and complete protease inhibitor mixture tablets (Roche Diagnostics) according to the manufacturer's instructions) on ice for 45 min. Nuclei were harvested by centrifugation at 5000 × g for 5 min and the remaining supernatant designated as the "cytoplasmic" fraction. The nuclear pellet was re-suspended in 0.3 ml of TKM buffer (50 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 25 mm KCl, 1 mm DTT, supplemented with protease and phosphatase inhibitors as described above), to which 0.6 ml of buffer D (80 mm Tris-HCl, pH 7.5, 2 mm EDTA, 530 mm NaCl, 1 mm DTT, supplemented with protease and phosphatase inhibitors as above) was added, before incubation on ice for 30 min. The nuclear extract was cleared by centrifugation at 14,000 rpm in a microcentrifuge at 4 °C, and used on the day of preparation. Co-immunoprecipitations—All steps were performed at 4 °C unless indicated otherwise. Nuclear and cytoplasmic extracts were made as described above. IHIC42 (anti-RAD51L3), IHIC48 (anti-XRCC2) (47Braybrooke J.P. Spink K.G. Thacker J. Hickson I.D. J. Biol. Chem. 2000; 275: 29100-29106Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), or appropriate preimmune sera were added to the extract at a dilution of 1:100, and the mixture was incubated on ice for 1 h. 100 μl of a 50% (w/v) slurry of protein A-Sepharose (in TKM/buffer D; ratio 1:2) was added, and the mixture was rotated end over end for 30 min at 4 °C. The immunoprecipitates were harvested in a microcentrifuge at 14,000 rpm for 5 s, and the pellet was washed with 1 ml of TKM/buffer D (ratio 1:2). The pellet was washed a further five times in the same buffer, before being boiled in protein loading buffer. Samples were separated by SDS-PAGE, transferred to nitrocellulose membrane, and Western blotted using conventional methods. GST "Pull-down" Experiments—Purified RAD51L3-GST or XRCC2-GST fusion proteins were bound to glutathione-agarose columns as described previously (46Wu L. Davies S.L. Levitt N.C. Hickson I.D. J. Biol. Chem. 2001; 276: 19375-19381Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 47Braybrooke J.P. Spink K.G. Thacker J. Hickson I.D. J. Biol. Chem. 2000; 275: 29100-29106Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) using a typical bed volume of 0.5 ml. All constructs for expression of GST fusion proteins have been described previously (47Braybrooke J.P. Spink K.G. Thacker J. Hickson I.D. J. Biol. Chem. 2000; 275: 29100-29106Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 48Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9636-9644Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar), except for N1-RAD51L3-GST, which was generated using PCR to eliminate the first 120 codons of the RAD51L3 cDNA. Briefly, purified recombinant protein or nuclear extracts were loaded onto the column at 4 °C, and the resin was washed with 15 ml of TKM/buffer D. The column matrix was boiled in protein sample loading buffer and the eluted proteins were separated by SDS-PAGE before Western blotting. Far Western Blotting Analysis—Typically, 0.2–0.5 μg of each polypeptide was subjected to SDS-PAGE and transferred to Hybond C-extra nitrocellulose membranes using a TE 70 semi-dry transfer unit (Hoeffer). All subsequent steps were performed at 4 °C. Filters were immersed twice in denaturation buffer (6 m guanidine HCl in PBSA) for 10 min, and then incubated for 6 × 10 min in serial dilutions (1:1) of denaturation buffer with PBSA supplemented with 1 mm DTT. Filters were blocked in PBSA containing 10% (w/v) powdered low fat milk, 0.1% (v/v) Tween 20 for 30 min before being incubated with purified recombinant RAD51L3 protein (47Braybrooke J.P. Spink K.G. Thacker J. Hickson I.D. J. Biol. Chem. 2000; 275: 29100-29106Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) in PBSA supplemented with 0.25% (w/v) milk, 0.1% (v/v) Tween 20, 1 mm DTT, and 1 mm phenylmethylsulfonyl fluoride for 60 min. Filters were washed for 4 × 10 min in PBSA containing 0.25% (w/v) powdered low fat milk, 0.1% (v/v) Tween 20. The second wash contained 0.00001% (v/v) glutaraldehyde. Conventional Western analysis was then performed to detect the presence of RAD51L3 using IHIC42 as the primary antibody. An otherwise identically treated negative control blot was processed, but without any incubation with RAD51L3. Purification of the RAD51L3-XRCC2 Complex—BL21(DE3) bacteria were transformed simultaneously with pJB3.21 and pJB1.2, and transformants were selected on LB agar containing ampicillin and kanamycin. Cultures of the doubly transformed bacteria were grown in LB medium containing ampicillin and kanamycin at 37 °C to an A600 of 0.6 before addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 0.4 mm. After 2 h of incubation with shaking, the culture was cooled to 4 °C in iced water for 15 min, and the cells were harvested by centrifugation. The cell pellet was re-suspended in 25 mm Tris-HCl, pH 7.5, 250 mm NaCl (1 ml of buffer per 40 ml of original bacterial culture volume), and the suspension was frozen at -80 °C. Following thawing of the suspension, all procedures were carried out at 4 °C unless stated otherwise. 0.2% (v/v) Triton X-100 and complete EDTA-free protease inhibitor tablets (Roche Diagnostics) were added and the mixture was incubated on ice for 30 min. Phenylmethylsulfonyl fluoride was added to the final concentration of 1 mm immediately prior to lysis by sonication (4 × 15 s at maximum amplitude with cooling on ice between bursts). The lysate was cleared by centrifugation at 39,000 rpm for 25 min (70 Ti rotor; Beckman) and ammonium sulfate was added to a final concentration of 45% to precipitate proteins. Following incubation on ice for 30 min, precipitated proteins were harvested by centrifugation, the pellet was re-suspended in 25 mm Tris-HCl, pH 7.5, 100 mm NaCl, and the solution was dialyzed for 2 h against nickel binding buffer (25 mm Tris-HCl, pH 7.5, 450 mm NaCl, 10 mm imidazole). The solution was then subjected to nickel chelate chromatography using a Poros MC20 column with a bed volume of 1.7 ml and a BioCAD work station (Perceptive Biosytems). Prior to loading, the column was charged with several bed volumes of 100 mm NiSO4, saturated with 5 bed volumes of 1.5 m imidazole in 25 mm Tris-HCl, pH 7.5, 450 mm NaCl, and finally was equilibrated with 5 bed volumes of 10 mm imidazole in the same buffer. After loading of
Referência(s)