Potential Role for the BLM Helicase in Recombinational Repair via a Conserved Interaction with RAD51

Bloom's syndrome (BS) is an autosomal recessive disorder that predisposes individuals to a wide range of cancers. The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases. The precise role played by these enzymes in the cell remains to be determined. However, genome-wide hyper-recombination is a feature of many RecQ helicase-deficient cells. In eukaryotes, a central step in homologous recombination is catalyzed by the RAD51 protein. In response to agents that induce DNA double-strand breaks, RAD51 accumulates in nuclear foci that are thought to correspond to sites of recombinational repair. Here, we report that purified BLM and human RAD51 interact in vitroand in vivo, and that residues in the N- and C-terminal domains of BLM can independently mediate this interaction. Consistent with these observations, BLM localizes to a subset of RAD51 nuclear foci in normal human cells. Moreover, the number of BLM foci and the extent to which BLM and RAD51 foci co-localize increase in response to ionizing radiation. Nevertheless, the formation of RAD51 foci does not require functional BLM. Indeed, in untreated BS cells, an abnormally high proportion of the cells contain RAD51 nuclear foci. Exogenous expression of BLM markedly reduces the fraction of cells containing RAD51 foci. The interaction between BLM and RAD51 appears to have been evolutionarily conserved since the C-terminal domain of Sgs1, theSaccharomyces cerevisiae homologue of BLM, interacts with yeast Rad51. Furthermore, genetic analysis reveals that theSGS1 and RAD51 genes are epistatic indicating that they operate in a common pathway. Potential roles for BLM in the RAD51 recombinational repair pathway are discussed. Bloom's syndrome (BS) is an autosomal recessive disorder that predisposes individuals to a wide range of cancers. The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases. The precise role played by these enzymes in the cell remains to be determined. However, genome-wide hyper-recombination is a feature of many RecQ helicase-deficient cells. In eukaryotes, a central step in homologous recombination is catalyzed by the RAD51 protein. In response to agents that induce DNA double-strand breaks, RAD51 accumulates in nuclear foci that are thought to correspond to sites of recombinational repair. Here, we report that purified BLM and human RAD51 interact in vitroand in vivo, and that residues in the N- and C-terminal domains of BLM can independently mediate this interaction. Consistent with these observations, BLM localizes to a subset of RAD51 nuclear foci in normal human cells. Moreover, the number of BLM foci and the extent to which BLM and RAD51 foci co-localize increase in response to ionizing radiation. Nevertheless, the formation of RAD51 foci does not require functional BLM. Indeed, in untreated BS cells, an abnormally high proportion of the cells contain RAD51 nuclear foci. Exogenous expression of BLM markedly reduces the fraction of cells containing RAD51 foci. The interaction between BLM and RAD51 appears to have been evolutionarily conserved since the C-terminal domain of Sgs1, theSaccharomyces cerevisiae homologue of BLM, interacts with yeast Rad51. Furthermore, genetic analysis reveals that theSGS1 and RAD51 genes are epistatic indicating that they operate in a common pathway. Potential roles for BLM in the RAD51 recombinational repair pathway are discussed. Bloom's syndrome glutathione S-transferase hydroxyurea homologous recombination methyl methanesulfonate sister chromatid exchange double-strand break polymerase chain reaction bromodeoxyuridine Germline mutations in the BLM gene give rise to Bloom's syndrome (BS),1 a rare disorder associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated increase in the occurrence of a wide range of cancers (1German J. Medicine. 1993; 72: 393-406Crossref PubMed Scopus (460) Google Scholar). BLM encodes a 159-kDa protein that is a member of the RecQ family of DNA helicases (2Ellis 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 (1221) Google Scholar). This highly conserved family of proteins is required for the maintenance of genomic stability in all organisms (3Karow J.K. Wu L. Hickson I.D. Curr. Opin. Genet. Dev. 2000; 10: 32-38Crossref PubMed Scopus (160) Google Scholar). In humans, five RecQ helicases have been identified. In addition to BLM, mutations in two other genes encoding RecQ helicases in humans have been associated with disease conditions: WRN andRECQ4, being defective in Werner's syndrome and Rothmund-Thomson syndrome, respectively (4Yu 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 (1496) Google Scholar, 5Kitao S. Shimamoto A. Goto M. Miller R.W. Smithson W.A. Lindor N.M. Furuichi Y. Nat. Genet. 1999; 22: 82-84Crossref PubMed Scopus (579) Google Scholar). Werner's syndrome is primarily associated with premature aging, and Rothmund-Thomson syndrome with skin and skeletal abnormalities, but both disorders also give rise to an elevated incidence of cancers (6Shen J.C. Loeb L.A. Trends Genet. 2000; 16: 213-220Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 7Vennos E.M. James W.D. Dermatol. Clin. 1995; 13: 143-150Abstract Full Text PDF PubMed Google Scholar). All RecQ family members contain a catalytic helicase domain that comprises seven highly conserved motifs found in many DNA and RNA helicases (3Karow J.K. Wu L. Hickson I.D. Curr. Opin. Genet. Dev. 2000; 10: 32-38Crossref PubMed Scopus (160) Google Scholar). Outside of this helicase domain, the RecQ family proteins show little sequence conservation. In BLM, these non-conserved domains are located both N- and C-terminal to the helicase domain and comprise ∼650 and 450 amino acids, respectively. It is likely that these non-conserved domains are important in functionally differentiating the roles of the different RecQ helicases within the cell by either providing additional enzymatic functions, such as the exonuclease activity dependent upon the N-terminal domain of WRN (8Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (376) Google Scholar, 9Shen J.C. Gray M.D. Oshima J. Kamath-Loeb A.S. Fry M. Loeb L.A. J. Biol. Chem. 1998; 273: 34139-34144Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), or by mediating interactions with other proteins (10Wu 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 (284) Google Scholar). Cells from BS patients display genomic instability, the diagnostic feature being an increase in the frequency of sister chromatid exchanges (SCEs) (11German J. Crippa L.P. Bloom D. Chromosoma. 1974; 48: 361-366Crossref PubMed Scopus (109) Google Scholar). Sonoda et al. (12Sonoda E. Sasaki M.S. Morrison C. Yamaguchi-Iwai Y. Takata M. Takeda S. Mol. Cell. Biol. 1999; 19: 5166-5169Crossref PubMed Scopus (368) Google Scholar) recently demonstrated that SCE formation requires homologous recombination (HR). Moreover, chicken BLM −/−cells display elevated SCE levels that are partially dependent onRAD54 (13Wang W. Seki M. Narita Y. Sonoda E. Takeda S. Yamada K. Masuko T. Katada T. Enomoto T. EMBO J. 2000; 19: 3428-3435Crossref PubMed Scopus (126) Google Scholar). BLM therefore seems to function in the regulation of HR events during replication. Consistent with this notion, mutations in SGS1 orrqh1 + , the budding and fission yeast RecQ homologues, respectively, also give rise to excessive recombination events (14Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (617) Google Scholar, 15Watt P.M. Hickson I.D. Borts R.H. Louis E.J. Genetics. 1996; 144: 935-945Crossref PubMed Google Scholar, 16Stewart E. Chapman C.R. Al-Khodairy F. Carr A.M. Enoch T. EMBO J. 1997; 16: 2682-2692Crossref PubMed Scopus (329) Google Scholar). In rqh1 mutants, inhibition of DNA replication, in particular, stimulates this excessive recombination (16Stewart E. Chapman C.R. Al-Khodairy F. Carr A.M. Enoch T. EMBO J. 1997; 16: 2682-2692Crossref PubMed Scopus (329) Google Scholar). Further evidence of a role for RecQ helicases in HR comes from the finding that RecQ, Sgs1, BLM, and WRN can all disrupt four-way junctions, a structural mimic for the Holliday junction intermediate formed during HR (17Harmon F.G. Kowalczykowski S.C. Genes Dev. 1998; 12: 1134-1144Crossref PubMed Scopus (239) Google Scholar, 18Bennett R.J. Keck J.L. Wang J.C. J. Mol. Biol. 1999; 289: 235-248Crossref PubMed Scopus (114) Google Scholar, 19Karow 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 (423) Google Scholar, 20Constantinou 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 (337) Google Scholar). Moreover, both BLM and WRN can promote branch migration of Holliday junctions (19Karow 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 (423) Google Scholar, 20Constantinou 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 (337) Google Scholar). The general mechanism for repair of DNA double-strand breaks (DSBs) by HR has been conserved in evolution. The central step involves the pairing of the DSB with homologous sequences to facilitate the exchange of DNA strands. In bacteria, this process is mediated by RecA, which forms a nucleoprotein filament on single-stranded DNA formed as a result of exonucleolytic processing of the DSB to generate single-stranded DNA tails. This nucleoprotein filament facilitates the search for homologous sequences and provides a structure within which DNA strand exchange occurs. In eukaryotes, essentially the same reaction is performed by RAD51, which is structurally related to RecA (21Ogawa T., Yu, X. Shinohara A. Egelman E.H. Science. 1993; 259: 1896-1899Crossref PubMed Scopus (561) Google Scholar, 22Sung P. Science. 1994; 265: 1241-1243Crossref PubMed Scopus (756) Google Scholar, 23Benson F.E. Stasiak A. West S.C. EMBO J. 1994; 13: 5764-5771Crossref PubMed Scopus (398) Google Scholar, 24Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). In Saccharomyces cerevisiae, the RAD51gene is a member of the RAD52 epistasis group, andrad51 mutants display defects in mitotic and meiotic recombination and sensitivity to ionizing radiation, highlighting the key role that RAD51 plays in both HR and recombinational repair of DNA strand breaks (25Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar). Studies on RAD51 in higher eukaryotes have been hampered by the fact that mice with a targeted disruption of theRAD51 gene die early during embryogenesis (26Lim D.S. Hasty P. Mol. Cell. Biol. 1996; 16: 7133-7143Crossref PubMed Scopus (628) Google Scholar, 27Tsuzuki 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: 6236-62340Crossref PubMed Scopus (672) Google Scholar). However, studies with early mouse embryos suggest that loss of RAD51renders cells sensitive to ionizing radiation (26Lim D.S. Hasty P. Mol. Cell. Biol. 1996; 16: 7133-7143Crossref PubMed Scopus (628) Google Scholar). Additionally,RAD51 mutant chicken cells, maintained by expression of human RAD51 (hRAD51) under the control of a regulable promoter, accumulate chromosomal breaks following repression of hRAD51 synthesis (28Sonoda E. Sasaki M.S. Buerstedde J.M. Bezzubova O. Shinohara A. Ogawa H. Takata M. Yamaguchi-Iwai Y. Takeda S. EMBO J. 1998; 17: 598-608Crossref PubMed Scopus (705) Google Scholar). This suggests a role for hRAD51 in the repair of DNA breaks in undamaged cycling cells, most likely those arising during DNA replication. Treatment of mammalian cells with agents that induce DSBs, such as ionizing radiation, induces localization of Rad51 to nuclear foci that are believed to correspond to multiprotein complexes engaged in recombinational repair (29Haaf T. Golub E.I. Reddy G. Radding C.M. Ward D.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2298-2302Crossref PubMed Scopus (497) Google Scholar). However, the precise composition of these recombinational repair centers is unknown. In this study, we have examined the possibility that the Bloom's syndrome gene product functions in the RAD51 recombinational repair pathway given the defects in recombination displayed by BS cells. BLM and hRAD51 were found to directly interact in vitro and co-immunoprecipitate from nuclear extracts. Consistent with these data, BLM forms nuclear foci, a subset of which, co-localize with hRAD51. Furthermore, the degree to which these two proteins co-localize to nuclear foci increases in response to ionizing radiation. The interaction between BLM and hRAD51 appears to have been evolutionarily conserved since Sgs1 physically associates with yeast RAD51, and genetic analysis reveals that the genes encoding these two proteins are epistatic. The SV40-transformed normal human fibroblast cell line, WI-38 (obtained from ATCC), was used as a representative normal human cell line. The GM08505 cell line is an SV40-transformed fibroblast cell line from a BS patient (obtained from NIGMS, National Institutes of Health, Bethesda, MD) and contains a BLMhomozygous frameshift mutation at residue 739 resulting in premature truncation of the protein (2Ellis 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 (1221) Google Scholar). PSNF5 cells were derived from a clone of GM08505 cells stably transfected with pcDNA3/BLM. Western blotting using an anti-BLM antiserum, IHIC33 (10Wu 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 (284) Google Scholar), was used to confirm that BLM was stably expressed by this line. Functional complementation of the BS phenotype was assessed by SCE frequency analysis which showed that PSNF5 cells have a near-normal SCE frequency. Derivation and characterization of these cells will be described elsewhere. 2P. S. North, et al., manuscript in preparation. All three cell lines were routinely cultured in α-minimal essential medium supplemented with 10% fetal bovine serum. HeLa S3 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum. The Escherichia coliBL21(DE3) strain was obtained from New England Biolabs. Gene disruptions were done in the S. cerevisiae YP-1 strain (his4-R leu2 MATa-URA3-MATa ura3–52 ade2–101 lys2). TheSGS1 open reading frame was replaced with LYS2 as described previously (15Watt P.M. Hickson I.D. Borts R.H. Louis E.J. Genetics. 1996; 144: 935-945Crossref PubMed Google Scholar). The RAD51 open reading frame was replaced with LEU2. The two-hybrid screen was performed in EGY48 (MATa his3 trp1 ura3–52 lex(leu2)3a). The generation of pcDNA3/BLM and pYES/BLM-NC has been described previously (10Wu 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 (284) Google Scholar). The plasmid pGEX-4T1 (Amersham Pharmacia Biotech) was used for the expression of glutathioneS-transferase (GST) fusion peptides. Portions of theBLM cDNA that encode various N- and C-terminal fragments of BLM were amplified by PCR. Sense and antisense primers contained the seven terminal 5′ sense and 3′ antisense codons, respectively, of each desired fragment. The antisense primer had an additional in-frame antisense stop codon. EcoRI and XhoI sites were also engineered into the sense and antisense primers, respectively, to allow in-frame cloning of the PCR fragments into pGEX-4T-1. For the two-hybrid screen, the LexA-fusion DNA binding domain vector (pEG202), the β-galactosidase reporter plasmid (pSH1834) and the plasmid pJG45, which contained the HeLa cDNA activation library, were kind gifts from Dr. R. Brent. pLexA-MAX (30Zervos A.S. Gyuris J. Brent R. Cell. 1993; 72: 223-232Abstract Full Text PDF PubMed Scopus (666) Google Scholar) and pHM12 (kindly provided by Drs. R. Finley and R. Brent) are derived from pEG202 and contain the entire open reading frame of human MAX and 295 residues of Drosophila melanogaster Cdc2 kinase, respectively. Portions of theBLM or SGS1 cDNA that encode various C-terminal regions of each protein were amplified by PCR and cloned into pEG202. Sense and antisense primers contained the seven terminal 5′ sense and 3′ antisense codons, respectively, of each desired fragment. The antisense primer had an additional in-frame antisense stop codon. EcoRI and XhoI sites were also engineered into the sense and antisense primers, respectively, to allow in-frame cloning of the PCR fragments into pEG202. The entire S. cerevisiae RAD51 open reading frame was amplified by PCR from yeast genomic DNA and cloned directionally into pJG45 viaEcoRI and XhoI sites that were engineered into the sense and antisense PCR primers, respectively. Polyclonal (IHIC33) and monoclonal (BFL103) anti-BLM antibodies have been described previously (10Wu 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 (284) Google Scholar). Anti-Rad51 antiserum was a kind gift from Dr. Steve West (Imperial Cancer Research Fund, London, United Kingdom). Anti-bromodeoxyuridine (BrdUrd) antibodies were from Dako. Recombinant BLM and BLM-NC proteins were expressed in yeast from the expression plasmids pYES/BLM and pYES/BLM-NC (10Wu 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 (284) Google Scholar), respectively, and purified using nickel chelate affinity chromatography as previously described (31Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 30611-30614Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Purified recombinant hRAD51 was a kind gift from Dr. Steve West. GST fusion peptides were expressed in and purified from BL21 (DE3) cells as previously described (10Wu 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 (284) Google Scholar). Nuclear extracts were prepared as previously described (10Wu 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 (284) Google Scholar) from exponentially growing WI-38 cells or cells that had been arrested in 5 μg/ml aphidicolin for 14 h. The HeLa cDNA activation library in pJG45 was transformed into EGY48, which already contained pEG202/BLM (residues 966–1417) and pSH1834 using a modified method of Gietz et al. (32Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2899) Google Scholar). Transformants were selected for uracil, histidine, and tryptophan prototrophy on 2% glucose, before being replica-plated to 2% galactose, 1% raffinose plates containing 40 μg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal). Plasmids from β-galactosidase positive colonies were isolated and sequenced. Intracellular localization of BLM and hRAD51 was visualized using the methodologies described previously (10Wu 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 (284) Google Scholar) using BFL103 and anti-Rad51 antibodies, respectively, in exponentially growing cells or cells that had been irradiated with 10 Gy or cultured with 5 μg/ml aphidicolin for 14 h. Quantitation of nuclear foci was determined from 100–200 cells for each treatment. For the detection of sites of DNA synthesis and hRAD51, cells grown on coverslips were incubated for 10 min at 37 °C in 150 μm BrdUrd, fixed in 4% paraformaldehyde in 250 mm HEPES (pH 7.4) at 4 °C for 20 min, permeabilized with 0.1% Triton X-100 at 4 °C for 20 min, followed by three washes with phosphate-buffered saline. Cells were blocked in phosphate-buffered saline containing 10% fetal calf serum for 15 min and then incubated for 1 h with anti-RAD51 antibodies and for 30 min with fluorescein isothiocyanate-conjugated secondary antibody (Dako). Incorporated BrdUrd was detected by a second fixation step of 20 min at 4 °C in 4% paraformaldehyde in 250 mm HEPES (pH 7.4) followed by a 15-min incubation in 8% paraformaldehyde in 250 mm HEPES (pH 7.4). Cells were then incubated in 4m HCl for 15 min at 37 °C followed by five washes in phosphate-buffered saline. Incorporated BrdUrd was immunolabeled using mouse monoclonal antibodies against BrdUrd and anti-mouse IgG conjugated with Cy3 (Sigma). DNA was visualized by Hoechst staining. Typically, nuclear extracts prepared from 108 cells were used for each immunoprecipitation. Immunoprecipitations were carried out as described previously (10Wu 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 (284) Google Scholar) with the exception that anti-Rad51 immunoprecipitates were captured. IHIC33 anti-serum was used to detect the presence of BLM using conventional Western blotting techniques. This technique was performed as described previously (10Wu 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 (284) Google Scholar) using recombinant full-length hRAD51 and either full-length recombinant BLM, BLM-NC, or GST fusion peptides containing N- or C-terminal fragments of BLM. Bound hRAD51 was detected using anti-RAD51 antiserum. Serial dilutions of exponentially growing yeast cultures were spotted onto either YPD plates or YPD plates containing drug and incubated for 2–3 days at 30 °C. Alternatively, exponentially growing yeast cultures were diluted into pre-warmed YPD medium or YPD medium containing drug, and the cell number monitored, using a Coulter counter (Sysmex F-820), over a period of 6 h. Growth inhibition was determined by calculating the percentage difference in cell number between the culture in drug-containing medium versus the control culture in drug-free YPD. Experiments were performed a minimum of three times on two independent segregants of each strain. The helicase domain in BLM, in common with the human WRN and RECQ4 proteins, is flanked by relatively large N- and C-terminal domains (3Karow J.K. Wu L. Hickson I.D. Curr. Opin. Genet. Dev. 2000; 10: 32-38Crossref PubMed Scopus (160) Google Scholar) that are likely important in functionally distinguishing these larger members of the RecQ helicase family. To analyze the role of these domains in BLM, we performed a yeast two-hybrid screen of a HeLa cDNA library using the C-terminal domain (residues 966–1417) of BLM as bait. One positive clone, isolated independently three times, was found to encode hRAD51. The interaction appeared specific since hRAD51 did not interact with two nonspecific control bait proteins (Fig. 1 A). Further mapping revealed that the C-terminal 150 amino acids of BLM (residues 1267–1417) were sufficient to mediate an interaction with hRAD51. Since only a fragment of BLM had been used in the two-hybrid screen, and interactions using this system could in principle be mediated by adaptor proteins, we used Far Western analysis with purified recombinant BLM and hRAD51 to determine if the full-length proteins were able to interact directly with each other. Far Western analysis revealed that the full-length proteins could interact (Figs.1 B and 2 B) and that the interaction was specific since hRAD51 did not interact with either of two control proteins used in this assay, GST and MBP (Fig. 1 C and data not shown). Using affinity-purified GST fusion peptides containing various portions of the C-terminal domain of BLM, the final 100 residues, representing amino acids 1317–1417 of BLM, were identified as being sufficient to mediate an interaction with hRAD51 (Fig. 1 C), thus providing independent confirmation of the location of the hRAD51 interaction domain on BLM identified by the yeast two-hybrid system (Fig. 1 A).Figure 2A portion of the N-terminal domain of BLM binds to hRAD51. A, Far Western analysis of full-length purified hRAD51 and a GST fusion peptide containing residues 1–212 of BLM (GST-BLM). Left panel is a Coomassie Blue-stained gel showing the purified GST-BLM fusion peptide alongside GST alone, as indicated on the left. Middle andright panels are the same proteins as shown in the left panel transferred to nitrocellulose membranes and then probed with hRAD51 (+) or buffer alone (−), as indicated above. The membranes were then Western-blotted for the presence of bound hRAD51 using an anti-hRAD51 antibody. B, Full-length BLM but not a fragment of BLM representing residues 213–1266 binds to hRAD51. Far Western analysis of hRAD51 against full-length purified recombinant BLM and BLM-NC. Nitrocellulose membranes, to which BLM and BLM-NC were transferred following SDS-PAGE, were incubated with hRAD51 (right panel) as indicated above and then probed for the presence of bound hRAD51 by Western blotting using an anti-hRAD51 antibody. Left panel shows a Western blot of an identical membrane using anti-BLM antibodies and indicates the relative amounts of each protein used. The positions of BLM and BLM-NC on the membranes are indicated on the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the possibility of additional hRAD51 interaction domains on BLM, Far Western analysis was performed between hRAD51 and a GST fusion protein containing the first 212 amino acids of BLM. hRAD51 was also able to specifically interact with this protein (Fig.2 A), indicating that BLM contains at least two domains with which hRAD51 can independently associate with (Figs. 1 C and 2 A). In contrast to full-length BLM, a purified recombinant mutant protein, BLM-NC, which consists of residues 213–1266 of BLM and therefore lacks both the N- and C-terminal hRAD51 interaction domains on BLM, was unable to bind hRAD51. This suggests that no additional portions of BLM interact with hRAD51 (Fig. 2 B). In response to DNA DSBs generated by ionizing radiation, hRAD51 localizes to nuclear foci that are thought to correspond to sites of ongoing repair (TableI, part A) (29Haaf T. Golub E.I. Reddy G. Radding C.M. Ward D.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2298-2302Crossref PubMed Scopus (497) Google Scholar). BLM also forms nuclear foci in undamaged normal cells (Table I, part A) that have been shown previously to correspond to PML nuclear bodies (33Ishov A.M. Sotnikov A.G. Negorev D. Vladimirova O.V. Neff N. Kamitani T. Yeh E.T.H. Strauss III, J.F. Maul G.C. J. Cell Biol. 1999; 147: 221-233Crossref PubMed Scopus (683) Google Scholar, 34Zhong S. Hu P. Ye T.-Z. Stan R. Ellis N.A. Pandolfi P.P. Oncogene. 1999; 18: 7941-7947Crossref PubMed Scopus (194) Google Scholar, 35Yankiwski V. Marciniak R.A. Guarente L. Neff N.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5214-5219Crossref PubMed Scopus (167) Google Scholar). In an asynchronous population of WI-38 cells, a human fibroblast cell line derived from a normal individual, the proportion of cells containing BLM foci and the average number of BLM foci/cell was found to increase in response to ionizing radiation (Table I, part A) (Fig.3). We therefore examined the possibility that BLM might co-localize with hRAD51 foci in response to ionizing radiation. In a population of unirradiated WI-38 cells, 16% of the cells contained a mean of 2.1 co-localizing BLM and hRAD51 foci/nucleus (Fig. 3 and Table I). Three hours after 10 Gy of γ-irradiation, these figures increased significantly such that 24% of the population contained a mean of 4.7 co-localizing BLM and hRAD51 foci/nucleus (Fig.3 and Table I, part B). By 6 h following irradiation, the proportion of cells containing BLM and hRAD51 co-localizing nuclear foci had declined to almost the level seen in the untreated population (18%). However, the mean number of co-localizing nuclear foci (3.4) was still significantly higher than that in the untreated control population (Table I, part B).Table IQuantitation of the proportion of cells containing BLM and hRAD51 foci in untreated, irradiated, or aphidicolin-treated WI-38 cells (A) and quantitation of nuclear foci containing both BLM and hRAD51 in untreated, irradiated, or aphidicolin-treated WI-38 cells (B)A.Untreatedγ-IrradiationAphidicolin3 h6 hCells containing BLM foci45%78%65%83%Cells containing hRAD51 foci22%93%94%93%Mean no. of BLM foci/cell4.4 (0–29)17.2 (0–48)6.5 (0–26)9.3 (0–22)Mean no. of hRAD51 foci/cell10.1 (0–38)27.2 (0–70)19.0 (0–60)11.7 (0–80)B.Untreatedγ-IrradiationAphidicolin3 h6 hNo. of BLM and hRAD51 co-localizing foci/cell084%76%82%59%1–515%15%13%38%>51%9%5%3%Mean no. of BLM and