A Double Holliday Junction Dissolvasome Comprising BLM, Topoisomerase IIIα, and BLAP75
2006; Elsevier BV; Volume: 281; Issue: 20 Linguagem: Inglês
10.1074/jbc.c600051200
ISSN1083-351X
AutoresSteven Raynard, Wendy Bussen, Patrick Sung,
Tópico(s)Ubiquitin and proteasome pathways
ResumoBloom syndrome (BS), an autosomal recessive disorder, is marked by a high incidence of cancer early in life. Cells derived from BS patients are unstable genetically and exhibit frequent sister chromatid exchanges, reflective of homologous recombination (HR) deregulation. BLM, the RecQ-like helicase mutated in BS, is found in several cellular protein complexes, all of which contain topoisomerase IIIα (Topo IIIα) and a novel protein BLAP75. Here, using highly purified human proteins, we show that BLAP75 associates independently with both Topo IIIα and BLM. Even though BLM and Topo IIIα can dissolve the double Holliday junction (DHJ) to yield non-crossover recombinants (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar), under physiological conditions, DHJ dissolution becomes completely dependent on BLAP75. The effect of BLAP75 on BLM-Topo IIIα is highly specific, as it is not seen with the combination of Topo IIIα and Escherichia coli RecQ helicase or another human RecQ-like helicase WRN. Thus, BLM, Topo IIIα, and BLAP75 constitute a dissolvasome complex that processes HR intermediates to limit DNA crossover formation. This function of the BLM-Topo IIIα-BLAP75 dissolvasome is likely indispensable for genome maintenance and cancer avoidance. Bloom syndrome (BS), an autosomal recessive disorder, is marked by a high incidence of cancer early in life. Cells derived from BS patients are unstable genetically and exhibit frequent sister chromatid exchanges, reflective of homologous recombination (HR) deregulation. BLM, the RecQ-like helicase mutated in BS, is found in several cellular protein complexes, all of which contain topoisomerase IIIα (Topo IIIα) and a novel protein BLAP75. Here, using highly purified human proteins, we show that BLAP75 associates independently with both Topo IIIα and BLM. Even though BLM and Topo IIIα can dissolve the double Holliday junction (DHJ) to yield non-crossover recombinants (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar), under physiological conditions, DHJ dissolution becomes completely dependent on BLAP75. The effect of BLAP75 on BLM-Topo IIIα is highly specific, as it is not seen with the combination of Topo IIIα and Escherichia coli RecQ helicase or another human RecQ-like helicase WRN. Thus, BLM, Topo IIIα, and BLAP75 constitute a dissolvasome complex that processes HR intermediates to limit DNA crossover formation. This function of the BLM-Topo IIIα-BLAP75 dissolvasome is likely indispensable for genome maintenance and cancer avoidance. Cells from Bloom syndrome (BS) 3The abbreviations used are: BS, Bloom syndrome; Topo, topoisomerase; HR, homologous recombination; DHJ, double Holliday junction; GST, glutathione S-transferase; IPTG, isopropyl β-d-thiogalactopyranoside; DTT, dithiothreitol; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate). 3The abbreviations used are: BS, Bloom syndrome; Topo, topoisomerase; HR, homologous recombination; DHJ, double Holliday junction; GST, glutathione S-transferase; IPTG, isopropyl β-d-thiogalactopyranoside; DTT, dithiothreitol; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate). patients exhibit highly elevated levels of sister chromatid exchanges, indicative of an impairment of the ability to regulate crossover recombination. Consistent with this characteristic, BLM, the RecQ-like helicase mutated in BS, has been found to cooperate with the type IA topoisomerase Topo IIIα to resolve the homologous recombination (HR) intermediate that harbors a double Holliday junction (DHJ) into non-crossover recombinants. This DHJ dissolution activity of the BLM-Topo IIIα complex is thought to be critical for the suppression of DNA crossover formation in mitotic cells and cancer avoidance in humans (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar).BLAP75 (BLM-Associated Polypeptide, 75 kDa) was first identified by Meetei et al. (2Meetei A.R. Sechi S. Wallisch M. Yang D. Young M.K. Joenje H. Hoatlin M.E. Wang W. Mol. Cell. Biol. 2003; 23: 3417-3426Crossref PubMed Scopus (291) Google Scholar) as a component of several BLM-containing complexes immunoprecipitable from HeLa nuclear extracts. In a subsequent study, it was shown that small interfering RNA-mediated knockdown of BLAP75 causes a decrease in BLM and Topo IIIα protein levels in cells (3Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar). Importantly, BLAP75 depletion phenocopies the increased frequency of sister chromatid exchanges characteristic of BLM-deficient cells (3Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar). Taken together, the available evidence indicates that BLAP75 exists as a complex with BLM and Topo IIIα (henceforth referred to as the BTB complex), but the mechanistic details of this relationship remain elusive. For instance, whether BLAP75 associates directly with BLM, or through Topo IIIα, is unknown. More importantly, it is not clear whether BLAP75 influences the DHJ dissolution activity of BLM-Topo IIIα or, as suggested previously (3Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar), serves as a structural component to promote protein complex formation. In this study, we have carried out biochemical analyses to define the role of the BTB complex in DHJ dissolution.EXPERIMENTAL PROCEDURESExpression and Purification of the BLAP75 Protein—BLAP75 cDNA (from the American Type Culture Collection) was placed under the T7 promoter in the vector pGEX-6P1 (GE Healthcare), which adds a cleavable amino-terminal glutathione S-transferase (GST)-tag to the BLAP75 protein. A carboxyl-terminal (His)6-tag was subsequently added by site-directed mutagenesis to generate the plasmid pGEX-BLAP75-(His)6, which was introduced into Escherichia coli Rosetta (DE3) pLysS cells (Novagen). Protein expression was induced for 18 h at 16 °C with 0.2 mm IPTG. Cells were harvested by centrifugation and stored at –80 °C. All the subsequent steps were carried out at 4 °C. Extract from 50 g of cell paste was prepared by sonication in 250 ml of cell breakage buffer (50 mm Tris-HCl, pH 7.5, 20% sucrose, 0.5 mm EDTA, 600 mm KCl, 0.01% Igepal, 1 mm DTT, and the following protease inhibitors: aprotinin, chymostatin, leupeptin, and pepstatin A at 3 μg/ml each, and 1 mm phenylmethylsulfonyl fluoride). The extract was clarified by ultracentrifugation (100,000 × g for 90 min) and then treated with ammonium sulfate (0.24 g/ml). Precipitated proteins were harvested by centrifugation (12,000 × g for 30 min) and redissolved in 100 ml of K buffer (20 mm KH2PO4, pH 7.4, 10% glycerol, 0.5 mm EDTA, 0.01% Igepal, 1 mm DTT, and protease inhibitors) with 800 mm KCl, and mixed with 2 ml glutathione-Sepharose (GE Healthcare) for 2 h at 4 °C. After washing the matrix with 40 ml of K buffer containing 500 mm KCl, the bound proteins were eluted with 20 mm glutathione in K buffer containing 150 mm KCl. Peak fractions were pooled and loaded directly onto a 1 ml Source Q column (GE Healthcare), which was washed with 10 ml of K buffer containing 150 mm KCl and then eluted with a 12 ml gradient of 150–850 mm KCl in K buffer. The BLAP75 peak fractions (∼300–450 mm KCl) were pooled and mixed with 1 ml of nickel-nitrilotriacetate-agarose (Qiagen) for 2 h at 4 °C. The beads were washed with 20 ml of K buffer containing 300 mm KCl and 10 mm imidazole before eluting BLAP75 with a 12-ml gradient of 10–300 mm imidazole in K buffer containing 150 mm KCl. The peak fractions (∼160–210 mm imidazole) were pooled and loaded onto a 0.5-ml Mono Q column (GE Healthcare), which was washed with 5 ml K buffer containing 150 mm KCl and then eluted with a 6-ml gradient of 150–850 mm KCl in K buffer. Peak fractions (∼400–500 mm KCl) were pooled and concentrated to ∼2 mg/ml in a Centricon 30 device (Millipore). Approximately 500 μg of BLAP75 was obtained. The concentration of BLAP75 was determined by densitometric comparison of multiple loadings of the purified protein against known amounts of bovine serum albumin and β-galactosidase in a Coomassie Blue-stained polyacrylamide gel.Expression and Purification of Other Proteins—Human BLM protein tagged with a (His)6 affinity epitope at its carboxyl terminus was purified from yeast cells tailored to express this protein (4Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 30611-30614Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) using a multistep procedure, details of which will be described elsewhere. Human topoisomerase IIIα cDNA containing an amino-terminal (His)6 affinity tag was subcloned into the bacterial vector pRSFDuet-1 (Novagen). A multistep protocol (to be described elsewhere) was developed for the purification of Topo IIIα. Recombinant (His)6-tagged WRN protein was purified from a baculovirus/insect cell expression system using a multistep procedure modified from the published protocol (5Orren D.K. Brosh Jr., R.M. Nehlin J.O. Machwe A. Gray M.D. Bohr V.A. Nucleic Acids Res. 1999; 27: 3557-3566Crossref PubMed Scopus (107) Google Scholar); the details of this procedure will be described elsewhere. Purified E. coli RecQ protein was a gift from Stephen Kowalczykowski. Protein concentrations were determined as described above.BLM-Topo IIIα Co-immunoprecipitation—Purified BLM and Topo IIIα, 3 μg each, were incubated in 30 μl of K buffer containing 150 mm KCl at 4 °C for 10 min and then rabbit polyclonal anti-BLM antibody (0.4 μl of a 1:5 dilution of ab476, Abcam Ltd.) was added, followed by another 30-min incubation at 4 °C. To capture BLM and associated Topo IIIα, the reactions were incubated for 30 min with 10 μl of protein-G-coupled magnetic beads (Dynal Biotech) with gentle mixing. The beads were isolated using a magnet, and after washing the beads twice with 30 μl of K buffer containing 150 mm KCl, the bound proteins were eluted with 30 μl of SDS-PAGE sample loading buffer. The supernatant that contained unbound proteins, the wash, and the SDS eluate, 10 μl each, were subjected to 7.5% SDS-PAGE and staining with Coomassie Blue to visualize the proteins.GST Pulldown Assay—For GST-pulldown experiments, GST-tagged BLAP75 without the (His)6-tag (3 μg) was incubated with purified BLM, Topo IIIα, WRN, or E. coli RecQ, 3 μg each, in 30 μl of K buffer containing 100 mm KCl at 4 °C for 10 min. The protein solutions were mixed with 5 μl of glutathione-Sepharose beads (GE Healthcare) for an additional 30 min at 4 °C with gentle mixing. After washing the beads twice with 30 μl of K buffer containing 100 mm KCl, the bound proteins were eluted with 30 μl of SDS-PAGE sample loading buffer. The supernatant, wash, and SDS eluate, 10 μl each, were resolved by 10% SDS-PAGE and proteins were stained with Coomassie Blue. (His)6-tagged Topo IIIα was visualized via Western blotting using horseradish peroxidase-conjugated anti-histidine antibodies (Sigma).DHJ Dissolution Assay—The DHJ substrate was prepared by hybridizing and ligating two oligonucleotides, 32P-labeled B1 and unlabeled R1, as described previously (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar, 6Fu T.J. Tse-Dinh Y.C. Seeman N.C. J. Mol. Biol. 1994; 236: 91-105Crossref PubMed Scopus (65) Google Scholar). Following ligation, the reaction was mixed with an equal volume of 90% formamide in TAE buffer (30 mm Tris acetate, pH 7.4, 0.5 mm EDTA) containing bromphenol blue (0.08%), heated at 95 °C for 5 min to dissociate all the unligated oligonucleotides, and then resolved in an 8% polyacrylamide gel containing 20% formamide and 8 m urea in TAE buffer at 55 °C. The portion of the gel containing the DHJ was excised, and the DNA within was electroeluted into TAE buffer in a dialysis tubing. The DHJ substrate was concentrated in a Centricon YM-30 device (Millipore) and then filter dialyzed into TE (20 mm Tris-HCl, pH 7.4, 0.2 mm EDTA).The DHJ dissolution reaction (Fig. 3, A and B) was carried out in the following manner: various mixtures of BLM (10.5 nm), Topo IIIα (100 nm), and the indicated amounts of BLAP75 were incubated for 10 min on ice in 11.5 μlof reaction buffer (50 mm Tris-HCl, pH 7.8, 1 mm DTT, 0.8 mm MgCl2, 200 μg/ml bovine serum albumin, 2 mm ATP, 80 mm or higher levels of KCl, and an ATP regenerating system consisting of 10 mm creatine phosphate and 50 μg/ml creatine kinase) followed by the incorporation of the DHJ substrate (1.2 nm) in 1 μl. After a 5-min incubation at 37 °C, 2 μl of 10% SDS and 0.5 μlof proteinase K (10 μg/μl stock) were added to the reaction mixtures, followed by a 3-min incubation at 37 °C. The deproteinized reactions were mixed with an equal volume of sample loading buffer (20 mm Tris-HCl, pH 7.5, 50% glycerol, and 0.08% Orange G) containing 50% urea, incubated at 95 °C for 3 min, and then resolved in 8% denaturing gels, as above. In Fig. 3C, E. coli RecQ or WRN, 15 nm each, was incubated with Topo IIIα and/or BLAP75 as described for BLM above.RESULTS AND DISCUSSIONBLAP75 cDNA harboring a carboxyl-terminal (His)6-tag and a cleavable amino-terminal GST-tag was constructed and placed under an IPTG-inducible T7 promoter for expression in E. coli. Cells harboring the GST-BLAP75 expression plasmid were treated with IPTG to induce protein expression. Coomassie Blue staining of bacterial extracts revealed a ∼110-kDa protein species that was absent in uninduced cells (Fig. 1A). We confirmed that this protein was BLAP75 by immunoblot analysis with anti-GST antibodies (Fig. 1A). For BLAP75 purification, E. coli lysate was subjected to precipitation with ammonium sulfate, followed by two chromatographic fractionation steps and affinity purification using glutathione-Sepharose and Ni2+-nitrilotriacetate-agarose (Fig. 1, B and C). We could remove the NH2-terminal GST-tag from BLAP75 by treatment with the PreScission Protease (GE Healthcare). However, control experiments revealed no functional difference between the GST-cleaved and non-cleaved forms of BLAP75 (data not shown). Several independently purified BLAP75 preparations gave the same results in all the biochemical analyses. For protein-protein interaction and functional studies, we expressed and purified the human BLM, Topo IIIα, and WRN proteins and also obtained the E. coli RecQ protein from the Kowalczykowski group; the SDS-PAGE analysis of these protein factors is shown in Fig. 1D.FIGURE 1Expression and purification of BLAP75. A, extracts from E. coli cells harboring pGEX-BLAP75-(His)6 grown with or without IPTG were analyzed by SDS-PAGE or immunoblotting with anti-GST antibodies. B, the procedure devised for the purification of BLAP75. C, purified BLAP75 (1 μg) was analyzed by SDS-PAGE. D, purified BLM, Topo IIIα, E. coli RecQ, and WRN proteins, 1 μg each, were analyzed by SDS-PAGE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate the hierarchy of protein-protein interactions within the BTB complex, we utilized the purified proteins in a series of pulldown experiments. Previous studies had reported an association of BLM with Topo IIIα by far Western analysis (7Wu 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). To confirm this interaction, we mixed purified BLM and Topo IIIα and then subjected the reaction mixture to immunoprecipitation with anti-BLM antibodies. As control, Topo IIIα alone was subjected to the same immunoprecipitation treatment. As shown in Fig. 2A, Topo IIIα was efficiently co-precipitated by BLM (lane 6), demonstrating a direct interaction between these two proteins. Next, to determine whether BLAP75 physically interacts with BLM, GST-BLAP75 or GST was incubated with BLM, followed by treatment of the reaction mixtures with glutathione-Sepharose beads to capture any protein complex that might have formed. Fig. 2B shows efficient retention of BLM on the glutathione-Sepharose beads with GST-BLAP75 but not GST alone, thus revealing a BLM-BLAP75 complex. The BLM-BLAP75 interaction is specific, as GST-BLAP75 did not bind the related E. coli RecQ or human WRN protein (Fig. 2D). We next enquired whether BLAP75 also associates with Topo IIIα. For this experiment, we performed GST-mediated pull-down with purified (His)6-tagged Topo IIIα and a form of GST-BLAP75 that lacks the (His)6 tag. Because the GST-BLAP75 and (His)6-tagged Topo IIIα have the same gel mobility, the latter was visualized by immunoblotting with anti-histidine antibodies. The results revealed that BLAP75 binds Topo IIIα with avidity (Fig. 2C).FIGURE 2BLAP75 physically interacts with BLM and Topo IIIα. A, purified BLM was incubated with purified Topo IIIα, and the reaction mixture was subjected to immunoprecipitation with anti-BLM antibodies. The reaction supernatant (S), wash (W), and eluate (E) were analyzed by SDS-PAGE. B and C, GST-BLAP75 or GST alone was incubated with BLM (in B) or Topo IIIα (in C). Protein complexes were captured on glutathione-Sepharose beads, followed by SDS-PAGE (in B) or immunoblotting with anti-histidine antibodies (in C). D, GST-BLAP75 or GST alone was incubated with WRN or E. coli RecQ, followed by mixing with glutathione-Sepharose beads to capture any protein complex that might have formed. Analysis was by SDS-PAGE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)During HR, a DNA intermediate harboring two Holliday junctions is generated, and the resolution of this intermediate by a specialized nuclease called "resolvase" yields a mixture of crossover and non-crossover recombinants (8Symington L.S. Microbiol. Mol. Biol. Rev. 2002; 66 (and table of contents): 630-670Crossref PubMed Scopus (810) Google Scholar). In mitotic cells, the formation of crossovers is specifically suppressed in a BLM-dependent manner (9Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar). Previous work has shown that BLM in conjunction with Topo IIIα can catalyze the dissolution of a DHJ that mimics the HR intermediate with two Holliday junctions to generate exclusively non-crossover recombinants (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar). Given that BLAP75 binds BLM and Topo IIIα (Fig. 2), we surmised that BLAP75 may influence DHJ dissolution by BLM-Topo IIIα. To test this hypothesis, we constructed a DHJ substrate by annealing and ligating two oligonucleotides (32P-labeled B1 and unlabeled R1) according to published procedures (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar, 6Fu T.J. Tse-Dinh Y.C. Seeman N.C. J. Mol. Biol. 1994; 236: 91-105Crossref PubMed Scopus (65) Google Scholar). As shown in Fig. 3A, BLAP75 greatly stimulated DHJ dissolution by BLM and Topo IIIα. Specifically, while the combination of BLM and Topo IIIα was able to dissolve about 10% of the DHJ substrate, the inclusion of BLAP75 led to >80% dissolution. Moreover, this effect became even more pronounced (20–40-fold stimulation by BLAP75) upon inclusion of a physiological level of salt in the reaction (Fig. 3B). We have verified that neither E. coli RecQ nor human WRN protein is capable of DHJ dissolution with or without BLAP75 and Topo IIIα (Fig. 3C).Published results (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar) have shown that DHJ dissolution by BLM and Topo IIIα leads to the formation of non-crossover products. We used a biotinylated version of the R1 oligonucleotide for constructing the DHJ substrate and the analytical procedures developed by Wu and Hickson (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar) to test whether the BTB complex likewise generates non-crossover recombinants (Fig. 4, A–C). Briefly, DHJ dissolution in the non-crossover manner would untangle the 32P-labeled B1 oligonucleotide and the biotinylated R1 oligonucleotide, such that treatment with magnetic streptavidin beads would deplete the DHJ substrate but not the radiolabeled product (Fig. 4A). Fig. 4B shows that the radiolabeled product made by the BTB complex was not depleted by treatment with streptavidin magnetic beads, indicating that it was free 32P-labeled B1 oligonucleotide formed via a non-crossover mode of DHJ dissolution. We also employed restriction analysis to further verify the non-crossover configuration of the DHJ dissolution products. The radiolabeled product was susceptible to the restriction enzyme HhaI but resistant to RsaI (Fig. 4C), again indicative of a non-crossover mode of DHJ dissolution by the BTB complex (see Fig. 4A for explanation). Just as what was previously reported for the combination of BLM and Topo IIIα (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar), the DHJ dissolution activity of the BTB complex requires ATP hydrolysis by the BLM protein, as no product was observed upon substitution of ATP with the non-hydrolyzable (or slowly hydrolyzable) analogue AMP-PNP or ATPγS (data not shown).FIGURE 4Non-crossover products are formed by the BTB complex. A, a biotinylated DHJ substrate was used to test whether the products of BTB-mediated dissolution have a non-crossover configuration. B, streptavidin magnetic beads depleted the DHJ substrate (lanes 1 and 2) but not the dissolution product (lanes 3 and 4). When unreacted DHJ was mixed with the dissolution product, only the former was depleted by the streptavidin magnetic beads (lanes 5 and 6). C, radiolabeled dissolution product (lane 2) was digested by HhaI (lane 3) but was resistant to RsaI (lane 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Even though homologous recombination is an important DNA repair tool and helps prevent DNA replication fork demise (8Symington L.S. Microbiol. Mol. Biol. Rev. 2002; 66 (and table of contents): 630-670Crossref PubMed Scopus (810) Google Scholar, 10Michel B. Grompone G. Flores M.J. Bidnenko V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12783-12788Crossref PubMed Scopus (277) Google Scholar), aberrant or untimely HR events can have deleterious effects (11Gangloff S. Soustelle C. Fabre F. Nat. Genet. 2000; 25: 192-194Crossref PubMed Scopus (309) Google Scholar, 12Klein H.L. Genetics. 2001; 157: 557-565Crossref PubMed Google Scholar). Deregulated HR, manifested as greatly elevated levels of sister chromatid exchanges and interhomologue crossover recombination, is likely responsible for the frequent genomic rearrangements and loss of heterozygosity in BLM-deficient cells (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar, 9Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar). This compromised genomic integrity is believed to be the cause of early onset cancers in BS patients (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar, 9Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar). Recent studies have found additional protein factors potentially required for BLM protein activities (2Meetei A.R. Sechi S. Wallisch M. Yang D. Young M.K. Joenje H. Hoatlin M.E. Wang W. Mol. Cell. Biol. 2003; 23: 3417-3426Crossref PubMed Scopus (291) Google Scholar, 3Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar). Here we have provided evidence that the BLM-associated polypeptide, BLAP75, binds BLM and Topo IIIα to assemble the BTB complex. Since under physiological conditions DHJ dissolution by BLM-Topo IIIα becomes completely dependent on BLAP75, aside from being important for the stability of BLM and Topo IIIα (3Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar), this novel polypeptide also plays an important functional role in the processing of HR intermediates to limit DNA crossover formation in cells.Overall, our biochemical analyses with highly purified protein factors have unveiled a novel DHJ dissolvasome complex that controls a major pathway of HR regulation and genome maintenance, and they also provide important insights into the mechanistic underpinnings of this complex. The diverse nature of components in higher order ensembles that harbor the BTB dissolvasome (3Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar) alludes to the intricate mechanisms involved in HR regulation and related processes. For instance, association of the BTB complex with components of the Fanconi anemia protein complex (2Meetei A.R. Sechi S. Wallisch M. Yang D. Young M.K. Joenje H. Hoatlin M.E. Wang W. Mol. Cell. Biol. 2003; 23: 3417-3426Crossref PubMed Scopus (291) Google Scholar, 13Pichierri P. Franchitto A. Rosselli F. EMBO J. 2004; 23: 3154-3163Crossref PubMed Scopus (110) Google Scholar) may link the BTB-mediated HR regulatory mechanism to the elimination of DNA cross-links via HR (14Kennedy R.D. D'Andrea A.D. Genes Dev. 2005; 19: 2925-2940Crossref PubMed Scopus (343) Google Scholar). Delineating the settings in which the BTB dissolvasome complex functions will be of great importance in enhancing our understanding of the significance of HR regulation in ensuring the fidelity of DNA repair reactions and in cancer avoidance. Our definition of the functional attributes of the BTB complex and the contributions of the individual components of this complex represents an important step toward this goal. Cells from Bloom syndrome (BS) 3The abbreviations used are: BS, Bloom syndrome; Topo, topoisomerase; HR, homologous recombination; DHJ, double Holliday junction; GST, glutathione S-transferase; IPTG, isopropyl β-d-thiogalactopyranoside; DTT, dithiothreitol; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate). 3The abbreviations used are: BS, Bloom syndrome; Topo, topoisomerase; HR, homologous recombination; DHJ, double Holliday junction; GST, glutathione S-transferase; IPTG, isopropyl β-d-thiogalactopyranoside; DTT, dithiothreitol; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate). patients exhibit highly elevated levels of sister chromatid exchanges, indicative of an impairment of the ability to regulate crossover recombination. Consistent with this characteristic, BLM, the RecQ-like helicase mutated in BS, has been found to cooperate with the type IA topoisomerase Topo IIIα to resolve the homologous recombination (HR) intermediate that harbors a double Holliday junction (DHJ) into non-crossover recombinants. This DHJ dissolution activity of the BLM-Topo IIIα complex is thought to be critical for the suppression of DNA crossover formation in mitotic cells and cancer avoidance in humans (1Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar). BLAP75 (BLM-Associated Polypeptide, 75 kDa) was first identified by Meetei et al. (2Meetei A.R. Sechi S. Wallisch M. Yang D. Young M.K. Joenje H. Hoatlin M.E. Wang W. Mol. Cell. Biol. 2003; 23: 3417-3426Crossref PubMed Scopus (291) Google Scholar) as a component of several BLM-containing complexes immunoprecipitable from HeLa nuclear extracts. In a subsequent study, it was shown that small interfering RNA-mediated knockdown of BLAP75 causes a decrease in BLM and Topo IIIα protein levels in cells (3Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar). Importantly, BLAP75 depletion phenocopies the increased frequency of sister chromatid exchanges characteristic of BLM-deficient cells (3Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar). Taken together, the available evidence indicates that BLAP75 exists as a complex with BLM and Topo IIIα (henceforth referred to as the BTB complex), but the mechanistic details of this relationship remain elusive. For instance, whether BLAP75 associates directly with BLM, or through Topo IIIα, is unknown. More importantly, it is not clear whether BLAP75 influences the DHJ dissolution activity of BLM-Topo IIIα or, as suggested previously (3Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar), serves as a structural component to promote protein complex formation. In this study, we have carried out biochemical analyses to define the role of the BTB complex in DHJ dissolution. EXPERIMENTAL PROCEDURESExpression and Purification of the BLAP75 Protein—BLAP75 cDNA (from the American Type Culture Collection) was placed under the T7 promoter in the vector pGEX-6P1 (GE Healthcare), which adds a cleavable amino-terminal glutathione S-transferase (GST)-tag to the BLAP75 protein. A carboxyl-terminal (His)6-tag was subsequently added by site-directed mutagenesis to generate the plasmid pGEX-BLAP75-(His)6, which was introduced into Escherichia c
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