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• W2072015368 abstract "Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair. Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair. Bloom's syndrome Bloom's syndrome protein polymerase chain reaction in vitro coupled transcription/translation reaction polyacrylamide gel electrophoresis polyvinylidene difluoride Tris-buffered saline nuclear extract Mut L homolog Bloom's syndrome (BS)1is a rare autosomal recessive disorder characterized by immunodeficiency, short stature, male infertility, and an increased risk of a broad spectrum of cancers (1German J. Dermatol. Clin. 1995; 13: 7-18Abstract Full Text PDF PubMed Google Scholar). Cells isolated from BS individuals are characterized by cytogenetic abnormalities, with the hallmark feature of hyperrecombination between sister chromatids. BS chromosomes also display increased levels of breaks, translocations, quadriradial formations, and telomeric associations (2German J. Cancer Genet. Cytogenet. 1997; 93: 100-106Abstract Full Text PDF PubMed Scopus (241) Google Scholar).The gene mutated in BS was positionally cloned and namedBLM; it encodes a 1417-amino acid protein with strong homology to the Escherichia coli RecQ family of DNA and RNA helicases (3Ellis 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 (1204) Google Scholar). The E. coli RecQ helicase participates in homologous recombination and suppresses illegitimate recombination (4Hanada K. Ukita T. Kohno Y. Saito K. Kato J. Ikeda H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3860-3865Crossref PubMed Scopus (239) Google Scholar,5Harmon F.G. Kowalczykowski S.C. Genes Dev. 1998; 12: 1134-1144Crossref PubMed Scopus (235) Google Scholar). Other eukaryotic RecQ family members include Sgs1p fromSaccharomyces cerevisiae and Rqh1p fromSchizosaccharomyces pombe; loss of function of either of these helicases results in genomic instability (6Watt P.M. Hickson I.D. Borts R.H. Louis E. Genetics. 1996; 144: 935-945Crossref PubMed Google Scholar, 7Stewart E. Chapman C.R. Al-Khodairy F. Carr A.M. Enoch T. EMBO J. 1997; 16: 2682-2692Crossref PubMed Scopus (326) Google Scholar). Mutations in other human RecQ helicases result in the rare autosomal recessive disorders Werner's syndrome and Rothmund-Thomson syndrome, also characterized by chromosomal instability and cancer predisposition (8Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1479) Google Scholar,9Kitao S. Lindor N.M. Shiratori M. Furuichi Y. Shimamoto A. Genomics. 1999; 61: 268-276Crossref PubMed Scopus (143) Google Scholar).The BLM helicase unwinds duplex DNA from 3′ to 5′ in the presence of ATP (10Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 0611-0614Abstract Full Text Full Text PDF Scopus (327) Google Scholar, 11Neff N.F. Ellis N.A. Ye T.Z. Noonan J. Huang K. Sanz M. Proytcheva M. Mol. Biol. Cell. 1999; 10: 665-676Crossref PubMed Scopus (124) Google Scholar). It also selectively recognizes and promotes branch migration of Holliday junctions in vitro (12Karow 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 (419) Google Scholar). BLM can be found in a large protein complex in the nucleus with other proteins involved in DNA repair such as BRCA1, ATM, MLH1, MSH2, MSH6, and replication factor C (13Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (95) Google Scholar). However, direct interactions of BLM have only been demonstrated biochemically with replication protein A (RPA) and topoisomerase IIIα (14Brosh J. 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 (254) Google Scholar, 15Wu 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 (281) Google Scholar, 16Johnson F.B. Lombard D.B. Neff N.F. Mastrangelo M.A. Dewolf W. Ellis N.A. Marciniak R.A. Yin Y. Jaenisch R. Guarente L. Cancer Res. 2000; 60: 1162-1167PubMed Google Scholar). These experiments suggest that the BLM helicase interacts with a variety of nuclear proteins to perform functions in DNA replication, recombination, or repair.To understand the role of the BLM helicase in maintaining genomic stability, a yeast two-hybrid screen was used to identify proteins that directly interact with BLM; the DNA mismatch repair protein MLH1 was identified. Far Western analysis and immunoprecipitations confirmed the direct interaction of these two proteins through the C terminus of BLM.In vitro assays did not demonstrate a role for BLM, however, in DNA mismatch repair. These data suggest that the BLM helicase and MLH1 may function cooperatively in maintaining genomic stability independent of DNA mismatch repair.RESULTSTo identify protein partners of the BLM helicase, cDNA segments encoding the N terminus, helicase domain and C terminus were cloned into a GAL4-yeast two-hybrid vector and used to screen a human B-lymphocyte cDNA library (Fig.1 A). The C terminus of BLM, amino acids 1036–1417, identified five clones, two of which contained a full-length cDNA encoding the DNA mismatch repair protein MLH1 (Fig. 1 B).IVTT products were mixed and immunoprecipitated to investigate the interaction between BLM and MLH1. MLH1 is present when mixed with the C terminus of BLM and immunoprecipitated with an antibody specific for BLM (Fig. 2). The C terminus of BLM is detectable when mixed with MLH1 and immunoprecipitated with an MLH1-specific antibody. The N-terminal and helicase domains of the BLM helicase were unable to co-precipitate with MLH1 (data not shown). These data provide further evidence that BLM and MLH1 interact in vitro through the C terminus of BLM.Figure 2Immunoprecipitations of in vitrotranscribed and translated ( IVTT ) protein products demonstrate the interaction between the C terminus of BLM and MLH1. Labeled [35S]methionine IVTT protein mixes of BLM-C and MLH1 were immunoprecipitated with antibodies to BLM or MLH1.Lanes 1 and 2 represent input controls of 10 µl of IVTT-BLM-C or 10 µl of IVTT-MLH1, respectively. Lane 3contains 50 µl of IVTT-BLM-C immunoprecipitated with α -MLH1.Lane 4 contains 50 µl of IVTT-MLH1 immunoprecipitated with α -BLM. Lane 5 contains a mixture of 50 µl of IVTT-MLH1 and 50 µl of BLM-C immunoprecipitated with α -BLM. Lane 6 contains a mixture of 50 µl of IVTT-MLH1 and 50 µl of BLM-C immunoprecipitated with goat IgG. Lane 7 contains a mixture of 50 µl of IVTT-MLH1 and 50 µl of IVTT-BLM-C immunoprecipitated with α -MLH1. Lane 8 contains a mixture of 50 µl of IVTT-MLH1 and 50 µl of IVTT-BLM-C immunoprecipitated with mouse IgG. Samples were resolved by 10% SDS-PAGE and visualized by autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To study this in vitro interaction, lysates from insect cells that expressed full-length BLM were mixed with lysates from the human erythroleukemia cell line K562. Immunoprecipitation with an α-MLH1 antibody, but not nonspecific IgG, immunoprecipitated BLM (Fig.3 A). Conversely, immunoprecipitation with α-BLM antibody demonstrated that MLH1 is associated with BLM (Fig 3 B). As a positive control, BLM was immunoprecipitated with α-BLM and probed for the replication protein A (RPA) 70-kDa subunit, a known protein partner of the BLM helicase (14Brosh J. 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 (254) Google Scholar). RPA was detected in the α-BLM immunoprecipitation but not in the IgG control immunoprecipitates (Fig. 3 C).Figure 3Mixed lysate immunoprecipitation demonstrates the interaction between full-length BLM and MLH1 or RPA. Lysates from insect cells infected with baculovirus expressing full-length BLM were mixed with K562 nuclear extracts (NE). Immunoprecipitated proteins were resolved on a 10% SDS-PAGE and transferred to membrane. The first lane of each Western blot contains K562 NE and insect cell lysates infected with BLM baculovirus (BLM) as a control to show the size and specificity of α-BLM (Novus), α-MLH1 (PharMingen) and α-RPA (Santa Cruz) antibodies. In A, the second lane contains mixed lysates immunoprecipitated with an IgG control while thethird lane contains mixed lysates immunoprecipitated with α-MLH1. In B, the second lane contains K562 NE immunoprecipitated with goat IgG, the third land contains K562 NE immunoprecipitated with α-BLM (Santa Cruz), the fourth lane contains mixed lysates immunoprecipitated with goat IgG, and the fifth lane contains mixed lysates immunoprecipitated with α-BLM (Santa Cruz). In B, the Western blot was probed with α-MLH1. Fig. 3 C shows the same Western blot asB stripped and probed with α-RPA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Far Western assays were performed to confirm the in vitrointeraction of the C terminus of BLM and MLH1. Protein induction studies in bacteria show that the C terminus of BLM was expressed at high levels in E. coli but was only present in the insoluble fraction (data not shown). The C terminus of BLM was consequently isolated under denaturing conditions using nickel chromatography and then slowly dialyzed into buffer. Purified fractions were analyzed by SDS-PAGE followed by Coomassie staining to identify several fractions that were more than 90% pure. Far Western assays then confirmed that the C terminus of BLM and MLH1 interact in vitro. Briefly, the C terminus of BLM was bound to PVDF membrane and incubated in nuclear extracts; bound proteins were eluted. Western blot analysis with an α-MLH1 antibody detected a 90-kDa band (as well as some smaller degradation products) that co-migrates with both the lysate control and IVTT MLH1 (Fig. 4). Although high salt washes greatly diminished the interaction between the two proteins, membrane alone and membrane coated with a nonspecific protein, cytochrome c, did not bind MLH1 demonstrating the specificity of the BLM-MLH1 interaction. MLH1 was also expressedin vitro by IVTT and was capable of binding to the immobilized C terminus of BLM (Fig. 4).Figure 4Far Western assays demonstrate the interaction between the BLM-C terminus and MLH1. Lane 1contains 25 µg of K562 nuclear extract (NE) to show the size of MLH1. The sample in lane 2 is IVTT-MLH1, which includes 10 µl of the IVTT-MLH1 mixture used in these experiments.Lane 3 shows proteins bound to BLM-C protein following incubation in 25 µg of K562 NE. Lane 4 shows protein bound to BLM-C protein following incubation with 25 µg of K562 NE and a 500 mm NaCl wash. Lane 5 shows protein bound to BLM-C protein following incubation with 10 µl of IVTT-MLH1.Lane 6 shows protein bound to PVDF membrane blocked with 5% non-fat dry milk following incubation with 10 µl of IVTT-MLH1.Lane 7 shows protein bound to cytochrome cfollowing incubation with 10 µl of IVTT-MLH1. Samples were resolved by 10% SDS-PAGE and visualized by autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To test for in vivo interactions, K562 NE were used for immunoprecipitation with α-BLM or α-MLH1 antibodies. MLH1 was co-immunoprecipitated with BLM from K562 NE but was not present in the IgG control immunoprecipitates (Fig. 5). Endogenous BLM could not be detected when lysates were immunoprecipitated with antibodies that recognize MLH1. This may be due to the low expression levels of BLM in this cell line, its cell cycle-specific regulation, or the binding affinity of the BLM antibodies. It should be noted that Wang et al. have immunoprecipitated MLH1 and identified BLM, although the reverse experiment was not performed (13Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (95) Google Scholar).Figure 5BLM and MLH1 interact in vivo. K562 nuclear extracts were immunoprecipitated with α-BLM antibodies or a goat IgG control. Lane 1contains lysates from K562 NE showing the size of MLH1. Lane 2 contains an immunoprecipitation from the same extracts using a goat IgG as a negative control. Lane 3 contains an immunoprecipitation from the same extracts using an α-BLM antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Finally, the functional significance of the interaction between BLM and MLH1 was tested by examining the ability of BS cell extracts to carry out DNA mismatch repair by measuring their ability to repair mispaired substrates in vitro. M13mp2 DNA was used as a repair substrate and contained a covalently closed (+) strand and a (−) strand with a nick (to direct repair to this strand) located several hundred base pairs away from the mispair in the lacZαcomplementation gene. The (+) strand encodes one plaque phenotype (either colorless or blue) and the (−) strand encodes another plaque phenotype. If the unrepaired heteroduplex is introduced into anE. coli strain deficient in methyl-directed mismatch repair, plaques will have a mixed phenotype due to expression of both strands. However, repair in a repair-proficient human cell extract will reduce the percentage of mixed plaques and increase the ratio of the (+) strand phenotype relative to that of the (−) strand phenotype as the nick directs repair to the (−) strand.BS cell extracts repair a G-G mispair as efficiently as a HeLa cell extract, which is repair proficient (Fig.6 A). Repair is observed regardless of whether the nick is 3′ or 5′ to the mismatch, consistent with the bi-directional repair capability of the human mismatch repair system. The change in the ratio of blue to colorless plaques (Fig.6 B) indicates that repair is specific for the minus strand as directed by the nick in that strand. The BS extract also repairs substrates containing an A-C mismatch or either of two different unrepaired nucleotides. In contrast to these results, extracts of cell lines exhibiting microsatellite instability and having mutations in any of four mismatch repair genes (hMSH2, GTBP,MLH1 , or PMS2) are uniformly deficient in strand-specific mismatch repair (data not shown). These results suggest that BLM does not directly function in the mismatch repair pathway but may play crucial roles in the processing of heteroduplex formations during replication, recombination, or other types of DNA repair.Figure 6DNA mismatch repair activities of BS and HeLa cell extracts are equivalent. A, repair efficiency in percent of a G-G mispair at position 88 in thelacZα-complementation gene in extracts of BS or HeLa cells. B, the ratio of pure blue to pure colorless plaques from extracts of BS or HeLa cells. The results reflect counting more than 500 plaques/variable. In addition to the results shown, repair in an extract from BS cells was also observed for the following substrates, with the (+) strand listed first, the (−) strand listed next (where a dash indicates a missing nucleotide), and the position of the mismatch listed last: C*A at 87, 25% repair; *T at 91, 86% repair; C* at 132–136, 82% repair. These substrates all contained a 3′-nick at position −264.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONYeast two-hybrid screens with specific domains of the BLM helicase were used to identify putative protein partners of BLM. With the C terminus of the BLM helicase as bait, candidate proteins were identified including the human DNA mismatch repair protein MLH1.In vitro immunoprecipitations and far Western analysis demonstrated a direct interaction between MLH1 and the C terminus of BLM. Finally, mixed lysate immunoprecipitation with cells over-expressing full-length BLM and in vivoimmunoprecipitations confirmed the interaction. Our results demonstrate that MLH1 is a protein partner of the BLM helicase in addition to topoisomerase IIIα and replication protein A (14Brosh J. 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 (254) Google Scholar, 15Wu 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 (281) Google Scholar, 16Johnson F.B. Lombard D.B. Neff N.F. Mastrangelo M.A. Dewolf W. Ellis N.A. Marciniak R.A. Yin Y. Jaenisch R. Guarente L. Cancer Res. 2000; 60: 1162-1167PubMed Google Scholar). In vitro DNA mismatch repair assays were performed to determine whether the BLM helicase is necessary for DNA mismatch repair. Cell extracts lacking functional BLM helicase activity were competent for DNA mismatch repair. These data suggest that the BLM/MLH1 interaction may be important for specific DNA repair events independent of the repair of single DNA mismatches.The BLM helicase is implicated in DNA replication, recombination, and/or repair. The primary defect leading to hyperrecombination in BS cells may be due to improper processing of DNA intermediates at stalled replication forks during DNA synthesis (21Lonn U. Lonn S. Nylen U. Winblad G. German J. Cancer Res. 1990; 50: 3141-3145PubMed Google Scholar, 22Chakraverty R.K. Hickson I.D. Bioessays. 1999; 21: 286-294Crossref PubMed Scopus (196) Google Scholar, 23Frei C. Gasser S.M. J. Cell Sci. 2000; 113: 2641-2646Crossref PubMed Google Scholar). These stalled forks can result in double strand breaks if they are not correctly handled by cellular DNA repair machinery. It is unclear exactly how the BLM helicase resolves stalled replication forks, but double strand break repair via homologous or non-homologous recombination may be one explanation (24Wang 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 may also recognize loops of di- and tri-nucleotide repeats generated by DNA replication due to polymerase slippage or by single-stranded annealing events occurring during gene conversion (25Kirkpatrick D.T. Petes T.D. Nature. 1997; 387: 929-931Crossref PubMed Scopus (134) Google Scholar). BLM may untangle these loops for mismatch repair complexes to process them efficiently.Experiments using yeast mutants of sgs1, the S. cerevisiae homologue of BLM and WRN, and the mismatch repair gene msh2 may also provide clues to the functional significance of the BLM-MLH1 interaction (26Myung K. Datta A. Clark C. Kolodner R.D. Nat. Genet. 2001; 27: 113-116Crossref PubMed Scopus (266) Google Scholar). sgs 1 mutation does not affect base substitution or frameshift mutation rates but increases gross chromosomal rearrangements. The mutation of both sgs1 and msh2 synergistically increases gross chromosomal rearrangements as well as homeologous recombination rates, suggesting that these proteins act in overlapping pathways to maintain genomic stability. Complexes containing BLM, MLH1, MSH2, and MSH6 have been identified using mass spectrometry (13Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (95) Google Scholar) suggesting common functional roles for these proteins. MSH2-MSH6 could function by directing BLM via MLH1 to Holliday junctions (27Marsischky G.T. Lee S. Griffith J. Kolodner R.D. J. Biol. Chem. 1999; 274: 7200-7206Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) since BLM can resolve these structures in vitro (12Karow 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 (419) Google Scholar).MLH1-MLH3 and MSH4-MSH5 also play crucial roles in recombination, although the exact functions of these proteins are not well defined (28Wang T.F. Kleckner N. Hunter N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13914-13919Crossref PubMed Scopus (236) Google Scholar, 29Ross-Macdonald P. Roeder G.S. Cell. 1994; 79: 1069-1080Abstract Full Text PDF PubMed Scopus (323) Google Scholar). Complexed proteins like MLH1-MLH3 could act as sensors for recombination intermediates and possibly direct the BLM helicase to resolve these intermediates. Future studies will determine the mechanism by which mismatch repair complexes and the BLM helicase function to maintain genomic stability. Bloom's syndrome (BS)1is a rare autosomal recessive disorder characterized by immunodeficiency, short stature, male infertility, and an increased risk of a broad spectrum of cancers (1German J. Dermatol. Clin. 1995; 13: 7-18Abstract Full Text PDF PubMed Google Scholar). Cells isolated from BS individuals are characterized by cytogenetic abnormalities, with the hallmark feature of hyperrecombination between sister chromatids. BS chromosomes also display increased levels of breaks, translocations, quadriradial formations, and telomeric associations (2German J. Cancer Genet. Cytogenet. 1997; 93: 100-106Abstract Full Text PDF PubMed Scopus (241) Google Scholar). The gene mutated in BS was positionally cloned and namedBLM; it encodes a 1417-amino acid protein with strong homology to the Escherichia coli RecQ family of DNA and RNA helicases (3Ellis 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 (1204) Google Scholar). The E. coli RecQ helicase participates in homologous recombination and suppresses illegitimate recombination (4Hanada K. Ukita T. Kohno Y. Saito K. Kato J. Ikeda H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3860-3865Crossref PubMed Scopus (239) Google Scholar,5Harmon F.G. Kowalczykowski S.C. Genes Dev. 1998; 12: 1134-1144Crossref PubMed Scopus (235) Google Scholar). Other eukaryotic RecQ family members include Sgs1p fromSaccharomyces cerevisiae and Rqh1p fromSchizosaccharomyces pombe; loss of function of either of these helicases results in genomic instability (6Watt P.M. Hickson I.D. Borts R.H. Louis E. Genetics. 1996; 144: 935-945Crossref PubMed Google Scholar, 7Stewart E. Chapman C.R. Al-Khodairy F. Carr A.M. Enoch T. EMBO J. 1997; 16: 2682-2692Crossref PubMed Scopus (326) Google Scholar). Mutations in other human RecQ helicases result in the rare autosomal recessive disorders Werner's syndrome and Rothmund-Thomson syndrome, also characterized by chromosomal instability and cancer predisposition (8Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1479) Google Scholar,9Kitao S. Lindor N.M. Shiratori M. Furuichi Y. Shimamoto A. Genomics. 1999; 61: 268-276Crossref PubMed Scopus (143) Google Scholar). The BLM helicase unwinds duplex DNA from 3′ to 5′ in the presence of ATP (10Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 0611-0614Abstract Full Text Full Text PDF Scopus (327) Google Scholar, 11Neff N.F. Ellis N.A. Ye T.Z. Noonan J. Huang K. Sanz M. Proytcheva M. Mol. Biol. Cell. 1999; 10: 665-676Crossref PubMed Scopus (124) Google Scholar). It also selectively recognizes and promotes branch migration of Holliday junctions in vitro (12Karow 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 (419) Google Scholar). BLM can be found in a large protein complex in the nucleus with other proteins involved in DNA repair such as BRCA1, ATM, MLH1, MSH2, MSH6, and replication factor C (13Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (95) Google Scholar). However, direct interactions of BLM have only been demonstrated biochemically with replication protein A (RPA) and topoisomerase IIIα (14Brosh J. 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 (254) Google Scholar, 15Wu 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 (281) Google Scholar, 16Johnson F.B. Lombard D.B. Neff N.F. Mastrangelo M.A. Dewolf W. Ellis N.A. Marciniak R.A. Yin Y. Jaenisch R. Guarente L. Cancer Res. 2000; 60: 1162-1167PubMed Google Scholar). These experiments suggest that the BLM helicase interacts with a variety of nuclear proteins to perform functions in DNA replication, recombination, or repair. To understand the role of the BLM helicase in maintaining genomic stability, a yeast two-hybrid screen was used to identify proteins that directly interact with BLM; the DNA mismatch repair protein MLH1 was identified. Far Western analysis and immunoprecipitations confirmed the direct interaction of these two proteins through the C terminus of BLM.In vitro assays did not demonstrate a role for BLM, however, in DNA mismatch repair. These data suggest that the BLM helicase and MLH1 may function cooperatively in maintaining genomic stability independent of DNA mismatch repair. RESULTSTo identify protein partners of the BLM helicase, cDNA segments encoding the N terminus, helicase domain and C terminus were cloned into a GAL4-yeast two-hybrid vector and used to screen a human B-lymphocyte cDNA library (Fig.1 A). The C terminus of BLM, amino acids 1036–1417, identified five clones, two of which contained a full-length cDNA encoding the DNA mismatch repair protein MLH1 (Fig. 1 B).IVTT products were mixed and immunoprecipitated to investigate the interaction between BLM and MLH1. MLH1 is present when mixed with the C terminus of BLM and immunoprecipitated with an antibody specific for BLM (Fig. 2). The C terminus of BLM is detectable when mixed with MLH1 and immunoprecipitated with an MLH1-specific antibody. The N-terminal and helicase domains of the BLM helicase were unable to co-precipitate with MLH1 (data not shown). These data provide further evidence that BLM and MLH1 interact in vitro through the C terminus of BLM.To study this in vitro interaction, lysates from insect cells that expressed full-length BLM were mixed with lysates from the human erythroleukemia cell line K562. Immunoprecipitation with an α-MLH1 antibody, but not nonspecific IgG, immunoprecipitated BLM (Fig.3 A). Conversely, immunoprecipitation with α-BLM antibody demonstrated that MLH1 is associated with BLM (Fig 3 B). As a positive control, BLM was immunoprecipitated with α-BLM and probed for the replication protein A (RPA) 70-kDa subunit, a known protein partner of the BLM helicase (14Brosh J. 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 (254) Google Scholar). RPA was detected in the α-BLM immunoprecipitation but not in the IgG control immunoprecipitates (Fig. 3 C)." @default.
• W2072015368 created "2016-06-24" @default.
• W2072015368 creator A5022688838 @default.
• W2072015368 creator A5040551044 @default.
• W2072015368 creator A5054055369 @default.
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• W2072015368 date "2001-08-01" @default.
• W2072015368 modified "2023-10-11" @default.
• W2072015368 title "The Bloom's Syndrome Protein (BLM) Interacts with MLH1 but Is Not Required for DNA Mismatch Repair" @default.
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