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• W2072484882 abstract "In this report, we have analyzed the protein encoded by the murine Brca2 locus. We find that murine Brca2 shares multiple properties with human BRCA2 including its regulation during the cell cycle, localization to nuclear foci, and interaction with Brca1 and Rad51. Murine Brca2 stably interacts with human BRCA1, and the amino terminus of Brca2 is sufficient for this interaction. Exon 11 of murine Brca2 is required for its stable association with RAD51, whereas the carboxyl terminus of Brca2 is dispensable for this interaction. Finally, in contrast to human BRCA2, we demonstrate that carboxyl-terminal truncations of murine Brca2 localize to the nucleus. This finding may explain the apparent inconsistency between the cytoplasmic localization of carboxyl-terminal truncations of human BRCA2 and the hypomorphic phenotype of mice homozygous for similar carboxyl-terminal truncating mutations. In this report, we have analyzed the protein encoded by the murine Brca2 locus. We find that murine Brca2 shares multiple properties with human BRCA2 including its regulation during the cell cycle, localization to nuclear foci, and interaction with Brca1 and Rad51. Murine Brca2 stably interacts with human BRCA1, and the amino terminus of Brca2 is sufficient for this interaction. Exon 11 of murine Brca2 is required for its stable association with RAD51, whereas the carboxyl terminus of Brca2 is dispensable for this interaction. Finally, in contrast to human BRCA2, we demonstrate that carboxyl-terminal truncations of murine Brca2 localize to the nucleus. This finding may explain the apparent inconsistency between the cytoplasmic localization of carboxyl-terminal truncations of human BRCA2 and the hypomorphic phenotype of mice homozygous for similar carboxyl-terminal truncating mutations. glutathioneS-transferase polyacrylamide gel electrophoresis phosphate-buffered saline Women inheriting mutations in the BRCA2tumor-suppressor gene have up to an 84% lifetime risk of developing breast cancer (1Ford D. Easton D.F. Stratton M. Narod S. Goldgar D. Bishop D.T. Weber B. Lenoir G. Chang-Claude J. Sobol H. Teare M.D. Struewing J. Arason A. Scherneck S. Peto J. Rebbeck T.R. Tonin P. Neuhausen S. Barkardottir R. Eyfjord J. Lynch H. Ponder B.A. Gayther S.A. Zelada-Hedman M. et al.Am. J. Hum. Gen. 1998; 62: 676-689Abstract Full Text Full Text PDF PubMed Scopus (2507) Google Scholar), and these tumors account for ∼35% of inherited breast cancers in women (2Wooster R. Bignell G. Lancaster J. Swift S. Seal S. Mangion J. Collins N. Gregory S. Gumbs C. Micklem G. Nature. 1995; 378: 789-792Crossref PubMed Scopus (2975) Google Scholar). BRCA2 encodes a 3418-amino acid nuclear protein of a predicted molecular mass of 384 kDa. Most disease-causing BRCA2 alleles contain truncating mutations that result in deletion of the three characterized nuclear localization signals present at the extreme carboxyl terminus of BRCA2 (3Spain B.H. Larson C.J. Shihabuddin L.S. Gage F.H. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13920-13925Crossref PubMed Scopus (122) Google Scholar, 4Yano K. Morotomi K. Saito H. Kato M. Matsuo F. Miki Y. Bioch. Biophys. Res. Commun. 2000; 270: 171-175Crossref PubMed Scopus (48) Google Scholar). Because these signals are required for the nuclear localization of human BRCA2, it has been postulated that truncating alleles ofBRCA2 are functionally equivalent to null alleles of this tumor suppressor gene (3Spain B.H. Larson C.J. Shihabuddin L.S. Gage F.H. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13920-13925Crossref PubMed Scopus (122) Google Scholar). Though its exact cellular role remains unclear, a growing body of evidence indicates that BRCA2 is involved in DNA damage-response pathways shared with BRCA1 and RAD51. BRCA2, BRCA1, and RAD51 are each co-regulated with highest levels of expression occurring during the S and G2/M phases of the cell cycle, and these proteins co-localize to discrete foci within the nucleus (5Vaughn J.P. Cirisano F.D. Huper G. Berchuck A. Futreal P.A. Marks J.R. Iglehart J.D. Cancer Res. 1996; 56: 4590-4594PubMed Google Scholar, 6Bertwistle D. Swift S. Marston N.J. Jackson L.E. Crossland S. Crompton M.R. Marshall C.J. Ashworth A. Cancer Res. 1997; 57: 5485-5488PubMed Google Scholar, 7Vaughn J.P. Davis P.L. Jarboe M.D. Huper G. Evans A.C. Wiseman R.W. Berchuck A. Iglehart J.D. Futreal P.A. Marks J.R. Cell Growth Differ. 1996; 7: 711-715PubMed Google Scholar, 8Chen Y. Farmer A.A. Chen C.F. Jones D.C. Chen P.L. Lee W.H. Cancer Res. 1996; 56: 3168-3172PubMed Google Scholar, 9Ruffner H. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7138-7143Crossref PubMed Scopus (179) Google Scholar, 10Gudas J.M. Li T. Nguyen H. Jensen D. Rauscher III, F.J. Cowan K.H. Cell Growth Differ. 1996; 7: 717-723PubMed Google Scholar, 11Chen F. Nastasi A. Shen Z. Brenneman M. Crissman H. Chen D.J. Mutat. Res. 1997; 384: 205-211Crossref PubMed Scopus (105) Google Scholar). Furthermore, human BRCA2 has been shown to physically interact with both RAD51 (12Chen P.L. Chen C.F. Chen Y. Xiao J. Sharp Z.D. Lee W.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5287-5292Crossref PubMed Scopus (337) Google Scholar, 13Marmorstein L.Y. Ouchi T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13869-13874Crossref PubMed Scopus (240) Google Scholar, 14Wong A.K.C. Pero R. Ormonde P.A. Tavtigian S.V. Bartel P.L. J. Biol. Chem. 1997; 272: 31941-31944Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 15Katagiri T. Saito H. Shinohara A. Ogawa H. Kamada N. Nakamura Y. Miki Y. Genes Chromosomes Cancer. 1998; 21: 217-222Crossref PubMed Scopus (49) Google Scholar, 16Chen J.J. Silver D.P. Walpita D. Cantor S.B. Gazdar A.F. Tomlinson G. Couch F.J. Weber B.L. Ashley T. Livington D.M. Scully R. Mol. Cell. 1998; 2: 317-328Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar) and BRCA1 (16Chen J.J. Silver D.P. Walpita D. Cantor S.B. Gazdar A.F. Tomlinson G. Couch F.J. Weber B.L. Ashley T. Livington D.M. Scully R. Mol. Cell. 1998; 2: 317-328Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). Human BRCA2 binds to RAD51 via eight BRC repeats, each 30–80 amino acids in length, that are located within exon 11 of BRCA2 (17Bork P. Blomberg N. Nilges M. Nat. Genet. 1996; 13: 22-23Crossref PubMed Scopus (161) Google Scholar, 18Bignell G. Micklem G. Stratton M.R. Asworth A. Wooster R. Hum. Mol. Genet. 1997; 6: 53-58Crossref PubMed Scopus (157) Google Scholar). These repeats have been demonstrated by yeast two-hybrid analysis to be both necessary and sufficient for stable binding of human BRCA2 to RAD51 (12Chen P.L. Chen C.F. Chen Y. Xiao J. Sharp Z.D. Lee W.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5287-5292Crossref PubMed Scopus (337) Google Scholar, 14Wong A.K.C. Pero R. Ormonde P.A. Tavtigian S.V. Bartel P.L. J. Biol. Chem. 1997; 272: 31941-31944Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 15Katagiri T. Saito H. Shinohara A. Ogawa H. Kamada N. Nakamura Y. Miki Y. Genes Chromosomes Cancer. 1998; 21: 217-222Crossref PubMed Scopus (49) Google Scholar). The region(s) of BRCA2 that are required for binding to BRCA1 have been less clearly defined, though the carboxyl-terminal third of BRCA2 has been shown to be dispensable for this interaction (16Chen J.J. Silver D.P. Walpita D. Cantor S.B. Gazdar A.F. Tomlinson G. Couch F.J. Weber B.L. Ashley T. Livington D.M. Scully R. Mol. Cell. 1998; 2: 317-328Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). Nevertheless, despite the identification of BRCA2-RAD51 and BRCA2-BRCA1 protein-protein interactions, the contribution of these interactions to the tumor-suppressor functions of BRCA2 remains uncertain. Mice bearing homozygous mutations in Brca2 that yield truncations of all eight BRC repeats uniformly die in uterobetween embryonic day 6.5-8.5, with elevated levels of p53 and p21 (19Sharan S.K. Morimatsu M. Albrecht U. Lim D.S. Regel E. Dinh C. Sands A. Eichele G. Hasty P. Bradley A. Nature. 1997; 386: 804-810Crossref PubMed Scopus (921) Google Scholar, 20Suzuki A. de la Pompa J.L. Hakem R. Elia A. Yoshida R. Rong M. Nishina H. Chuang T. Wakeham A. Itie A. Koo W. Billia P. Ho A. Fukumoto M. Hui C.C. Mak T.W. Genes Dev. 1997; 11: 1242-1252Crossref PubMed Scopus (235) Google Scholar, 21Ludwig T. Chapman D.L. Papioannou V.E. Efstradiatis A. Genes Dev. 1997; 11: 1226-1241Crossref PubMed Scopus (463) Google Scholar). Notably, this phenotype is similar to that of mice homozygous for null mutations in either Rad51 orBrca1 (22Tsuzuki 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: 6236-6240Crossref PubMed Scopus (672) Google Scholar, 23Gowen L.C. Johnson B.L. Latour A.M. Sulik K.K. Koller B.H. Nat. Genet. 1996; 12: 191-194Crossref PubMed Scopus (397) Google Scholar, 24Hakem R. de la Pompa J.L. Sirard C. Mo R. Woo M. Hakem A. Wakeham A. Potter J. Reitmair A. Billia F. Firpo E. Hui C.C. Roberts J. Rossant J. Mak T.W. Cell. 1996; 85: 1009-1023Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 25Lim D.S. Hasty P. Mol. Cell. Biol. 1996; 16: 7133-7143Crossref PubMed Scopus (628) Google Scholar, 26Hakem R. de la Pompa J.L. Elia A. Potter J. Mak T.W. Nat. Genet. 1997; 16: 298-302Crossref PubMed Scopus (228) Google Scholar). Whereas mice bearing truncating alleles ofBrca2 that remove only a subset of BRC repeats also die in utero, a fraction of homozygotes survive to birth with the survival rate being roughly proportional to the number of BRC repeats left intact (27Connor F. Bertwistle D. Mee P.J. Ross G.M. Swift S. Grigorieva E. Tybulewicz V.L.J. Ashworth A. Nat. Genet. 1997; 17: 423-430Crossref PubMed Scopus (365) Google Scholar, 28Friedman L.S. Thistlewaite F.C. Patel K.J., Yu, V.P.C.C. Lee H. Venkitaraman A.R. Abel K.J. Carlton M.B.L. Hunter S.M. Coledge W.H. Evans M.J. Ponder B.A.J. Cancer Res. 1998; 58: 1338-1343PubMed Google Scholar, 29Patel K., Yu, V.P.C.C. Lee H. Corcoran A. Thistlewaite F.C. Evans M.J. Colledge W.H. Friedman L.S. Ponder B.A.J. Venkitaraman A.R. Mol. Cell. 1998; 1: 347-357Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). Surviving homozygotes invariably succumb to thymic lymphomas, and cells from these mice exhibit increased genotoxin sensitivity and chromosomal instability, as well as an impaired ability to form Rad51 nuclear foci in response to DNA damage (27Connor F. Bertwistle D. Mee P.J. Ross G.M. Swift S. Grigorieva E. Tybulewicz V.L.J. Ashworth A. Nat. Genet. 1997; 17: 423-430Crossref PubMed Scopus (365) Google Scholar, 28Friedman L.S. Thistlewaite F.C. Patel K.J., Yu, V.P.C.C. Lee H. Venkitaraman A.R. Abel K.J. Carlton M.B.L. Hunter S.M. Coledge W.H. Evans M.J. Ponder B.A.J. Cancer Res. 1998; 58: 1338-1343PubMed Google Scholar, 29Patel K., Yu, V.P.C.C. Lee H. Corcoran A. Thistlewaite F.C. Evans M.J. Colledge W.H. Friedman L.S. Ponder B.A.J. Venkitaraman A.R. Mol. Cell. 1998; 1: 347-357Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 30Yu V.P. Koehler M. Steinlein C. Schmid M. Hanakahi L.A. van Gool A.J. West S.C. Venkitaraman A.R. Genes Dev. 2000; 14: 1400-1406PubMed Google Scholar). In contrast, mice homozygous for truncating mutations inBrca2 that leave exon 11 intact exhibit a more limited sensitivity to genotoxins, are 100% viable, and do not appear to develop spontaneous tumors (31Morimatsu M. Donoho G. Hasty P. Cancer Res. 1998; 58: 3441-3447PubMed Google Scholar). These data argue for a central role of exon 11 in the genomic surveillance and tumor-suppressor functions of Brca2. Whereas murine knockout models support a role for BRCA2 as a tumor suppressor, the increasingly severe defects observed in mice as larger amounts of the Brca2 carboxyl terminus are truncated appear inconsistent with reports that even small carboxyl-terminal truncations in human BRCA2 result in its cytoplasmic localization. That is, essentially all truncating alleles might be expected to behave similarly to null alleles, because carboxyl-terminal truncation would ostensibly lead to cytoplasmic localization and preclude Brca2 from participating in nuclear functions (3Spain B.H. Larson C.J. Shihabuddin L.S. Gage F.H. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13920-13925Crossref PubMed Scopus (122) Google Scholar, 4Yano K. Morotomi K. Saito H. Kato M. Matsuo F. Miki Y. Bioch. Biophys. Res. Commun. 2000; 270: 171-175Crossref PubMed Scopus (48) Google Scholar). This apparent discrepancy could be because of differences in the subcellular localization signals of human and murine Brca2 or to differences in the functions of murine and human BRCA2 in the cytoplasm. In this regard, another apparent functional difference between murine and human BRCA2 is suggested by the mapping of a murine Brca2-Rad51 interaction to the carboxyl terminus of murine Brca2, because similar approaches have shown that the corresponding region of human BRCA2 lacks significant affinity for RAD51 (12Chen P.L. Chen C.F. Chen Y. Xiao J. Sharp Z.D. Lee W.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5287-5292Crossref PubMed Scopus (337) Google Scholar, 14Wong A.K.C. Pero R. Ormonde P.A. Tavtigian S.V. Bartel P.L. J. Biol. Chem. 1997; 272: 31941-31944Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 15Katagiri T. Saito H. Shinohara A. Ogawa H. Kamada N. Nakamura Y. Miki Y. Genes Chromosomes Cancer. 1998; 21: 217-222Crossref PubMed Scopus (49) Google Scholar). Further complicating the direct comparison of murine and human BRCA2 is the fact that the overall amino acid homology between these orthologs is only 59%, a relatively low degree of evolutionary conservation compared with other tumor-suppressor genes. Together, these data have called into question the applicability of murine models for understanding the function of human BRCA2. In this report, we characterize the murine Brca2 protein. We find that Brca2 stably interacts with murine Brca1 and Rad51. We demonstrate that the physical association of Brca2 with Rad51 requires exon 11 of murine Brca2 but not its carboxyl terminus. We also show that murine Brca2 differs from human BRCA2 in that carboxyl-terminal truncations of murine Brca2 localize to the nucleus. Collectively, our findings suggest that multiple functional interactions of Brca2 have been evolutionarily conserved with the notable exception of those signals required for its nuclear localization. Poly(A)+ RNA isolated from day 14 murine embryos was used to generate a cDNA library in lambda ZAP using the ZAP-cDNA synthesis and ZAP-cDNA Gigapack II Gold packaging kits according to manufacturer's instructions (Stratagene). 5 × 105 plaques from each library were screened by standard methods using [32P]dCTP-labeled random-primed cDNA fragments (BMB) corresponding to nucleotides 2–221, 798–2932, and 9033–9972 of murine Brca2. Hybridization was performed at a concentration of 106 cpm/ml in 48% formamide, 10% dextran sulfate, 4.8× SSC, 20 mm Tris, pH 7.5, 10× Denhardt's solution, 20 μg/ml salmon sperm DNA, and 0.1% SDS at 42 °C overnight. Filters were washed twice in 2× SSC/0.1% SDS at room temperature for 20 min and twice in 0.2× SSC/0.1% SDS for 20 min at 50 °C and subjected to autoradiography on XAR-5 film (Eastman Kodak Co.). Phage clones were plaque purified, and plasmids were liberated by in vivo excision according to the manufacturer's instructions. Sequence analysis identified three overlapping clones that together spanned the entire Brca2 coding sequence, with the exception of an internal deletion of nucleotides 454–672. This region was replaced with a polymerase chain reaction product generated from murine testis first-strand cDNA and primers 5′-GAATTCATGCCCGTTGAATACAAAAGGAGAC-3′ and 5′-CTCGAGGCAGATTTCCTCATTCTGGCTG-3′. After sequencing to verify the absence of additional mutations, the overlapping clones were assembled to generate a full-length murine Brca2 cDNA. Using primers 5′-CATCCGAATTCTGCAGCACAGCGATTTAGGAC-3′ and 5′-CATCCCTCGAGGCACCGCAGAGTAAGAGGG-3′ (Brca2A), and 5′-CATCCGAATTCGATGAAGAAGCACGCAGCTC-3′ and 5′-CATCCCTCGAGACTGCATTTTTCACAGTGGC-3′ (Brca2B), polymerase chain reaction products corresponding to amino acids 19 to 135 (Brca2A) and 206 to 566 (Brca2B) were generated from a partial Brca2cDNA, ligated into pGEM-T vector (Promega), and subcloned in-frame into pGEX-6P-1 (Amersham Pharmacia Biotech). GST1 fusion peptides were purified from BL21 Escherichia coli according to manufacturer's instructions. Brca2 peptides were cleaved from the GST domain using a site-specific protease, gel-purified by SDS-PAGE, and injected into rabbits using standard immunization protocols (Cocalico Biologicals). Sera from immunized rabbits were affinity-purified on columns containing immunogen bound to cyanogen bromide-activated Sepharose (Amersham Pharmacia Biotech) according to published methods (32Harlow E. Lane D. Antibodies: A Laboratory Manual. 1st Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988: 313-318Google Scholar). 293T cells were transiently transfected using a standard calcium-phosphate protocol (33Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2nd Ed. John Wiley & Sons, Inc., New York2000: 9.1.4-9.1.6Google Scholar). For co-immunoprecipitation experiments, 2.5 × 106 cells on 150-mm dishes were transfected with 25 μg of DNA. For subcellular localization studies, 1 × 106 cells on 100-mm dishes were transfected with 5 μg DNA, and cells were split onto culture slides at 24 h post-transfection. All analyses were performed at 48 h post-transfection. All cells were grown at 37 °C in a humidified incubator supplemented with 5% CO2. 293T, NMuMG, and 16MB9A cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (Gem Cell), 2 mm l-glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin. HC11 cells were cultured in RPMI supplemented with 10% bovine calf serum (Gem Cell), 1 mml-glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, 10 ng/ml epidermal growth factor, and 5 μg/ml insulin. Cells were harvested by lysis in EBC Buffer (50 mm Tris, pH 8.0, 120 mm NaCl, 0.5% Igepal CA-630 (Sigma)), supplemented with phosphatase inhibitors (50 mm NaF and 1 mmβ-glycerol phosphate) and protease inhibitors (100 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pefabloc). Following removal of insoluble debris by centrifugation, extracts were either boiled in 1× (final) Laemmli sample buffer (2% SDS, 10% glycerol, 60 mm Tris, pH 6.8, 5% β-mercaptoethanol, 250 mm dithiothreitol, 0.005% bromphenol blue) or subjected to immunoprecipitation. For immunoprecipitation studies, 1.0–1.5 mg of protein extract was incubated with 4 μg of affinity-purified antibody or, in the case of RAD51, 1 μl of polyclonal antiserum (Ab-1; Oncogene Science) for 1 h at 4 °C in a total volume of 1 ml of EBC plus inhibitors. Protein A-Sepharose (25 μl of a 50% slurry in PBS; Life Technologies, Inc.) was added, and incubation was continued for 1 h. The Sepharose beads were pelleted and washed three times in EBC plus inhibitors, resuspended, boiled in 14 μl of 2× (final) Laemmli sample buffer, and loaded for SDS-PAGE. Except as noted, all protein samples were separated by 5% SDS-PAGE in 50 mm Tris base, 192 mm glycine, and 0.1% SDS. For immunoblotting, electrophoresed proteins were transferred onto nitrocellulose (Schleicher & Schuell) in 50 mm Tris base, 192 mm glycine, and 20% methanol in a submerged tank apparatus (Bio-Rad) for 12 h at 20 V. Blotted membranes were rinsed twice in PBS and blocked for 1 h at room temperature in PBS containing 5% nonfat milk and 0.05% Igepal CA-630 (MPBS-I). All affinity-purified rabbit polyclonal primary antibodies were used for immunoblotting at a final concentration of 2 μg/ml. Commercial antibodies, including anti-human BRCA2 Ab-2 (Oncogene Science), anti-RAD51 Ab-1 (Oncogene Science), anti-RAD51 Ab-1 (NeoMarkers), anti-BRCA1 MS110 (Oncogene Science), anti-β-tubulin N357 (Amersham Pharmacia Biotech), and anti-RAD50 R75020 (Transduction Laboratories) were used at the concentrations recommended by the manufacturer. Blots were incubated with primary antibodies diluted in MPBS-I for 1 h at room temperature and were subsequently washed three times in MPBS-I for 10 min each. Peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Jackson Immunoresearch) were incubated at a 1:5000 dilution for 1 h at room temperature in MPBS-I. Blots were washed three times in MPBS-I for 15 min each and rinsed four times with PBS, and antibody complexes were detected by chemiluminescence (ECL Plus; Amersham Pharmacia Biotech) on XAR-5 film (Kodak). HC11 cells were synchronized by serum starvation for 48 h and were subsequently restimulated with growth medium containing 20% serum. At 4-h intervals, cells were trypsinized and washed in PBS, and approximately two-thirds of cells were pelleted and snap-frozen for subsequent protein harvest. The remaining cells were pelleted, resuspended in PBS, and fixed in 70% ethanol. Following fixation, cells were pelleted, resuspended in PBS supplemented with 10 μg/ml propidium iodide and 100 μg/ml RNase A, and sorted by DNA content using a Becton Dickinson FACScan flow cytometer. The program ModFit was used to quantify percentages of cells in each phase of the cell cycle. Nuclear and cytoplasmic fractionation was performed as described previously (34Wilson C.A. Ramos L. Villasenor M.R. Anders K.H. Press M.F. Clarke K. Karlan B. Chen J.J. Scully R. Livingston D. Zuch R.H. Kanter M.H. Cohen S. Calzone F.J. Slamon D.J. Nat. Genet. 1999; 21: 236-240Crossref PubMed Scopus (372) Google Scholar). Briefly, 16MB9A cells were harvested by trypsinization, pelleted, and washed in PBS. Cells were washed in ice-cold hypotonic buffer (30 mmHEPES, pH 7.5, 5 mm KCl, 1 mmMgCl2), resuspended in three packed cellular volumes of hypotonic buffer supplemented with protease inhibitors, and incubated on ice for 30 min. Cells were homogenized in a Wheaton Dounce with 25 strokes of a type B pestle. An equal volume of Nonidet P-40 lysis buffer (0.1% Igepal CA-630, 250 mm sucrose, 1 mm MgCl2, 10 mm Tris, pH 7.5) was added dropwise, and cells were lysed using another 10 strokes. Nuclei were pelleted at 1300 × g at 4 °C for 5 min. Following removal of the cytoplasmic supernatant, nuclei were washed twice in 1:1 hypotonic buffer/Nonidet P-40 lysis buffer and resuspended in an amount of 1:1 hypotonic buffer/Nonidet P-40 lysis buffer equal to the extract volume prior to centrifugation of nuclei. Nuclear and cytoplasmic fractions were diluted with 6× EBC to a final concentration of 1×, centrifuged to remove insoluble debris, and boiled in 1× (final) Laemmli sample buffer prior to SDS-PAGE. Cells were cultured in 2-well culture slides (Falcon), rinsed in PBS, and fixed for 10 min in 3% paraformaldehyde/2% sucrose/PBS. Cells were rinsed twice in PBS and permeabilized for 5 min in ice-cold buffer (0.5% Triton, 20 mm HEPES, pH 7.4, 50 mm NaCl, 3 mmMgCl2, 300 mm sucrose). Following five rinses in PBS, cells were incubated at 37 °C for 20 min with anti-Brca2A (2 μg/ml in 3% bovine serum albumin/PBS). Cells were rinsed twice in PBS and stained with a 1:200 dilution of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson Immunoresearch) in 3% bovine serum albumin/PBS. Stained cells were rinsed three times in PBS, mounted in Vectastain (Vector Laboratories), and visualized using a Bio-Rad MRC-1024 confocal microscope with a Kr/Ar laser. For subcellular localization of Brca2 isoforms, tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit (Jackson Immunoresearch) was used as a secondary antibody at a 1:200 dilution, and cells were visualized using confocal microscopy as above. To investigate the function of the murine Brca2 protein, we raised antisera to two recombinant Brca2 polypeptides corresponding to amino acid residues 19–135 and 206–566. Sera from these rabbits were affinity-purified and designated anti-Brca2A and anti-Brca2B, respectively. To demonstrate that these antibodies specifically recognize murine Brca2, we transfected 293T cells with either a full-length cDNA encodingBrca2 or an empty vector control. Immunoblotting of whole cell extracts with anti-Brca2A demonstrated a band of the predicted molecular mass in 293T cells transfected with a Brca2cDNA but not in cells transfected with a control vector (Fig.1A). This band co-migrates with endogenous Brca2 detected in the mammary epithelial cell lines HC11, NMuMG, and 16MB9A. Endogenous human BRCA2, detected in 293T cells using an antibody recognizing a carboxyl-terminal epitope of BRCA2, also co-migrates with this band (Fig. 1A). In contrast, no co-migrating band is detected by immunoblot of whole cell extracts from CAPAN-1 cells, in which a 6174dT frameshift mutation results in a carboxyl-terminal truncation of the BRCA2 gene product (Fig.1A). Detection of murine Brca2 was completely blocked by pre-incubating anti-Brca2A with the cognate GST-Brca2 (residues 19–135) fusion protein but not by pre-incubating with GST (data not shown). These findings indicate that anti-Brca2A specifically detects murine Brca2. As further evidence of the specificity of anti-Brca2A, we performed reciprocal immunoprecipitations and immunoblots on murine mammary epithelial cell extracts with anti-Brca2A and anti-Brca2B. As shown in Fig. 1B, anti-Brca2A detects a polypeptide of the appropriate molecular mass present in cell extracts subjected to immunoprecipitation with anti-Brca2B but not with control rabbit IgG. Similarly, anti-Brca2B detects a polypeptide of the appropriate molecular mass in cell extracts subjected to immunoprecipitation with anti-Brca2A. Moreover, the polypeptide immunoprecipitated by anti-Brca2A and anti-Brca2B co-migrates with the polypeptides detected by these antibodies in whole cell extracts (see below, and see Fig. 4). In aggregate, these data demonstrate that anti-Brca2A and anti-Brca2B specifically recognize murine Brca2. Our laboratory has demonstrated previously that steady-state levels of murine Brca2 mRNA are regulated during the cell cycle with peak expression occurring near the G1/S transition (35Rajan J.V. Wang M. Marquis S.T. Chodosh L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13078-13083Crossref PubMed Scopus (204) Google Scholar). Human BRCA2 has been shown to exhibit a similar pattern of regulation at both the RNA and protein levels (5Vaughn J.P. Cirisano F.D. Huper G. Berchuck A. Futreal P.A. Marks J.R. Iglehart J.D. Cancer Res. 1996; 56: 4590-4594PubMed Google Scholar, 6Bertwistle D. Swift S. Marston N.J. Jackson L.E. Crossland S. Crompton M.R. Marshall C.J. Ashworth A. Cancer Res. 1997; 57: 5485-5488PubMed Google Scholar). To determine whether steady-state levels of murine Brca2 protein are similarly regulated, HC11 cells were synchronized by serum starvation for 48 h, restimulated with serum, and harvested at 4-h intervals. Immunoblotting revealed that murine Brca2 expression is up-regulated beginning at ∼12 h following restimulation with serum, which coincides with cellular entry into S phase as shown both by flow cytometry and up-regulation of cyclin A (Fig.2). This up-regulation persists as cells exit S phase and enter the G2/M phase of the cell cycle (Fig. 2). Similar results were observed in a second mammary epithelial cell line, NMuMG (data not shown). These findings indicate that the cell cycle regulation of BRCA2 is conserved between mouse and human at both the RNA and protein level. Human BRCA2 has been shown to localize to nuclei by biochemical subcellular fractionation (6Bertwistle D. Swift S. Marston N.J. Jackson L.E. Crossland S. Crompton M.R. Marshall C.J. Ashworth A. Cancer Res. 1997; 57: 5485-5488PubMed Google Scholar). To determine whether murine Brca2 shares this property, nuclear and cytoplasmic fractions were prepared from the murine mammary epithelial cell line, 16MB9A. The purity of these fractions was confirmed by immunoblotting for β-tubulin and RAD50 as controls for cytoplasmic and nuclear proteins, respectively (Fig.3A). This analysis demonstrated that murine Brca2 is found primarily in the nuclear fraction of 16MB9A cells (Fig. 3A). Consistent with the nuclear localization of human BRCA2, immunofluorescence studies have shown that this protein localizes to subnuclear foci (16Chen J.J. Silver D.P. Walpita D. Cantor S.B. Gazdar A.F. Tomlinson G. Couch F.J. Weber B.L. Ashley T. Livington D.M. Scully R. Mol. Cell. 1998; 2: 317-328Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). To determine whether murine Brca2 exhibits a similar localization, we performed indirect immunofluorescence on NMuMG cells using anti-Brca2A. Numerous nuclear foci were observed in cells stained with anti-Brca2A but not in cells stained with a secondary antibody alone (Fig. 3B). Fluorescent signal was completely blocked by the cognate GST-Brca2 (residues 19–135) fusion protein but not by GST alone (data not shown). Collectively, these data demonstrate that, similar to human BRCA2, murine Brca2 is a nuclear protein and localizes to discrete foci in mammary epithelial cell lines. Human BRCA2 has been demonstrated previously to physically interact with the homology-based DNA repair protein, RAD51 (12Chen P.L. Chen C.F. Chen Y. Xiao J. Sharp Z.D. Lee W.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5287-5292Crossref PubMed Scopus (337) Google Scholar, 13Marmorstein L.Y. Ouchi T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13869-13874Crossref PubMed Scopus (240) Google Scholar, 14Wong A.K.C. Pero R. Ormonde P.A. Tavtigian S.V. Bartel P.L. J. Biol. Chem. 1997; 272: 31941-31944Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 15Katagiri T. Saito H. Shinohara A. Ogawa H. Kamada N. Nakamura Y. Miki Y. G" @default.
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