The RING Heterodimer BRCA1-BARD1 Is a Ubiquitin Ligase Inactivated by a Breast Cancer-derived Mutation

Abstract

BRCA1-BARD1 constitutes a heterodimeric RING finger complex associated through its N-terminal regions. Here we demonstrate that the BRCA1-BARD1 heterodimeric RING finger complex contains significant ubiquitin ligase activity that can be disrupted by a breast cancer-derived RING finger mutation in BRCA1. Whereas individually BRCA1 and BARD1 have very low ubiquitin ligase activities in vitro, BRCA1 combined with BARD1 exhibits dramatically higher activity. Bacterially purified RING finger domains comprising residues 1–304 of BRCA1 and residues 25–189 of BARD1 are capable of polymerizing ubiquitin. The steady-state level of transfected BRCA1 in vivo was increased by co-transfection of BARD1, and reciprocally that of transfected BARD1 was increased by BRCA1 in a dose-dependent manner. The breast cancer-derived BARD1-interaction-deficient mutant, BRCA1C61G, does not exhibit ubiquitin ligase activity in vitro. These results suggest that the BRCA1-BARD1 complex contains a ubiquitin ligase activity that is important in prevention of breast and ovarian cancer development. BRCA1-BARD1 constitutes a heterodimeric RING finger complex associated through its N-terminal regions. Here we demonstrate that the BRCA1-BARD1 heterodimeric RING finger complex contains significant ubiquitin ligase activity that can be disrupted by a breast cancer-derived RING finger mutation in BRCA1. Whereas individually BRCA1 and BARD1 have very low ubiquitin ligase activities in vitro, BRCA1 combined with BARD1 exhibits dramatically higher activity. Bacterially purified RING finger domains comprising residues 1–304 of BRCA1 and residues 25–189 of BARD1 are capable of polymerizing ubiquitin. The steady-state level of transfected BRCA1 in vivo was increased by co-transfection of BARD1, and reciprocally that of transfected BARD1 was increased by BRCA1 in a dose-dependent manner. The breast cancer-derived BARD1-interaction-deficient mutant, BRCA1C61G, does not exhibit ubiquitin ligase activity in vitro. These results suggest that the BRCA1-BARD1 complex contains a ubiquitin ligase activity that is important in prevention of breast and ovarian cancer development. ubiquitin ubiquitin-activating enzyme ubiquitin carrier protein SKP1-CUL1 (CDC53)- F-box protein complex anaphase-promoting complex polyacrylamide gel electrophoresis, GST, glutathioneS-transferase hemagglutinin dithiothreitol isopropyl-1-thio-β-d-galactopyranoside Germline mutations of BRCA1 predispose women to breast and ovarian cancers (1Zheng L. Li S. Boyer T.G. Lee W.H. Oncogene. 2000; 19: 6159-6175Crossref PubMed Scopus (136) Google Scholar). BRCA1 contains several domains that interact with a variety of molecules and is potentially responsible for multiple functions in DNA damage repair, transcription, and cell-cycle regulation (2Venkitaraman A.R. Science. 1999; 286: 1100-1102Crossref PubMed Scopus (48) Google Scholar, 3Haile D.T. Parvin J.D. J. Biol. Chem. 1999; 274: 2113-2117Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 4Xu X. Weaver Z. Linke S.P. Li C. Gotay J. Wang X.W. Harris C.C. Ried T. Deng C.X. Mol. Cell. 1999; 3: 389-395Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). BARD1 was identified in a yeast two-hybrid screen as a protein that interacts with BRCA1 (5Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.C. Hwang L.Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (626) Google Scholar). Both BRCA1 and BARD1 proteins contain a RING finger (5Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.C. Hwang L.Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (626) Google Scholar) and exist as homodimers or preferentially form stable heterodimers (6Meza J.E. Brzovic P.S. King M-C. Klevit R.E. J. Biol. Chem. 1999; 274: 5659-5665Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The heterodimeric interaction is mediated by the flanking regions of the RING finger motif of the two molecules (6Meza J.E. Brzovic P.S. King M-C. Klevit R.E. J. Biol. Chem. 1999; 274: 5659-5665Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Although a transcriptional function in the C terminus of BRCA1 has been recently reported (3Haile D.T. Parvin J.D. J. Biol. Chem. 1999; 274: 2113-2117Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), the biochemical function of the heterodimeric RING finger constituted from the N termini of BRCA1 and BARD1 is not known. Previously, we and others identified a highly conserved small RING finger protein, ROC1 (also called Rbx1 and Hrt1), as an essential subunit of the SCF Ub1 ligase (7Ohta T. Michel J.J. Schottelius A.J. Xiong Y. Mol. Cell. 1999; 3: 535-541Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 8Tan P. Fuchs S.Y. Chen A. Wu K. Gomez C. Ronai Z. Pan Z.Q. Mol. Cell. 1999; 3: 527-533Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 9Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin Jr., W.G. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Science. 1999; 284: 657-661Crossref PubMed Scopus (670) Google Scholar, 10Seol J.H. Feldman R.M. Zachariae W. Shevchenko A. Correll C.C. Lyapina S. Chi Y. Galova M. Claypool J. Sandmeyer S. Nasmyth K. Deshaies R.J. Shevchenko A. Deshaies R.J. Genes Dev. 1999; 13: 1614-1626Crossref PubMed Scopus (360) Google Scholar). The Ub ligase (E3) catalyzes the formation of polyubiquitin chains onto substrate proteins via isopeptide bonds utilizing the Ubs that have been sequentially activated by enzymes E1 and E2. Polyubiquitinated substrates are then rapidly degraded by the 26 S proteasome (11Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). The SCF and the APC are the two major Ub ligase complexes that regulate ubiquitin-mediated proteolysis during G1/S and anaphase (12Peters J.M. Curr. Opin. Cell Biol. 1998; 10: 759-768Crossref PubMed Scopus (225) Google Scholar), and contain the small RING finger proteins ROC1 and APC11, respectively (7Ohta T. Michel J.J. Schottelius A.J. Xiong Y. Mol. Cell. 1999; 3: 535-541Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 8Tan P. Fuchs S.Y. Chen A. Wu K. Gomez C. Ronai Z. Pan Z.Q. Mol. Cell. 1999; 3: 527-533Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 9Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin Jr., W.G. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Science. 1999; 284: 657-661Crossref PubMed Scopus (670) Google Scholar, 10Seol J.H. Feldman R.M. Zachariae W. Shevchenko A. Correll C.C. Lyapina S. Chi Y. Galova M. Claypool J. Sandmeyer S. Nasmyth K. Deshaies R.J. Shevchenko A. Deshaies R.J. Genes Dev. 1999; 13: 1614-1626Crossref PubMed Scopus (360) Google Scholar). Point mutations in the RING finger domain of ROC1 completely disrupted the Ub ligase activity, suggesting an essential role of the domain for its activity (7Ohta T. Michel J.J. Schottelius A.J. Xiong Y. Mol. Cell. 1999; 3: 535-541Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). APC11 also contains Ub ligase activity in vitro (7Ohta T. Michel J.J. Schottelius A.J. Xiong Y. Mol. Cell. 1999; 3: 535-541Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). More recently, several large RING finger proteins, such as MDM2, c-Cbl, IAP, and AO7, with otherwise diverse structures and functions were linked to ubiquitination (13Honda R. Yasuda H. Oncogene. 2000; 19: 1473-1476Crossref PubMed Scopus (315) Google Scholar, 14Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (916) Google Scholar, 15Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (873) Google Scholar, 16Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (947) Google Scholar), suggesting a potentially broad and general function for RING fingers in activating Ub ligase activity. One of these RING proteins, BRCA1, has been closely scrutinized for Ub ligase activity. However, the ability of BRCA1 by itself to promote ubiquitin polymerization was found to be limited (16Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (947) Google Scholar). In this report, we have provided evidence demonstrating that the RING finger of BRCA1, in concert with BARD1, exhibits significant ubiquitin ligase activity. This activity can be disrupted by a breast cancer-derived RING finger mutation of BRCA1, suggesting a direct relationship between the ubiquitin ligase function of BRCA1 and breast cancer development. cDNAs for full-length human BARD1 and CstF-50 were amplified by polymerase chain reaction from a Hela cell cDNA library with pfu polymerase (Stratagene) and subcloned into the pcDNA3 vector inframe with appropriate tags. pGEX-BARD1-(1–111) was created by digesting the full-length BARD1 with HindIII, and subcloning into the pGEX-KG vector. pET-His6-BARD1-(14–189) and pET-His6-BARD1-(25–189) were created by digesting the full-length BARD1 with BamHI and PstI, andNcoI and PstI, respectively and subcloned into the pET-3E-His6 vector. Full-length BRCA1 cDNA was a generous gift from Dr. Wen-Hwa Lee. The N-terminal fragment (1) of BRCA1 was generated by digesting the full-length BRCA1 at itsKpnI restriction site and subcloning into either the pGEX vector or the pcDNA3 vector with the appropriate tag. pGEX-BRCA1-(1–342) was created by self-ligation ofBglII-digested pGEX-BRCA1-(1–772). pET-His6-BRCA1-(1–304) was created by digesting BRCA1-(1–772) with EcoRI and subcloning into the pET-3E-His6 vector. BRCA1 or BARD1 point mutations were introduced by site-directed mutagenesis (Stratagene). cDNA for each human E2/Ubc was amplified by polymerase chain reaction from a HeLa cell cDNA library and subcloned into the pET-3E-His6vector. cDNA for human E2F1 and cyclin B1 in the pcDNA3 expression vector were gifts from Dr. Yue Xiong. All the constructs used were verified by DNA sequencing. Cells (293T) were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10#x0025; fetal bovine serum (Life Technologies, Inc.) and 1% antibiotic-antimycotic agent (Life Technologies, Inc.) in a 37 #x00B0;C incubator with 5#x0025; CO2. DNA was transfected using the standard calcium phosphate precipitation method. For each transfection, the total plasmid DNA was adjusted to 15 μg per 100-mm dish by adding the parental pcDNA3 vector. For immunoprecipitation, cells were harvested 36 h after transfection and lysed by incubating at 4 °C for 1 h with 0.6 ml per 100 mm dish of buffer A containing 15 mm Tris-HCl pH 7.5, 0.5m NaCl, 0.35#x0025; Nonidet P-40, 1 mmphenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 10 μg/ml trypsin inhibitor, and 150 μg/ml benzamidine. Lysed cells were then clarified by centrifugation at 100,000 ×g at 4 °C for 1 h. The supernatants (0.3 ml) were mixed with 3 μg of anti-Myc (9E10) or anti-HA (12CA5) antibody, and then the antibody-bound proteins were precipitated with protein A-agarose beads (7.5 μl). The proteins bound to the beads were used either for the Ub ligation assay or immunoblotting. For straight immunoblotting to analyze the steady-state levels of Myc-BRCA1 or HA-BARD1, the transfected cells were lysed and clarified as described above, and 50 μg of each sample was resolved by SDS-PAGE on a 7.5% gel, followed by immunoblotting. Rabbit E1 was purchased from Affiniti Research Products. His6-tagged Ub with a protein kinase C recognition site, His6-tagged E2 proteins, and His6-E2F1 were purified as previously described (7Ohta T. Michel J.J. Schottelius A.J. Xiong Y. Mol. Cell. 1999; 3: 535-541Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 17Ohta T. Xiong Y. Cancer Res. 2001; 61: 1347-1353PubMed Google Scholar). His6-BARD1-(14–189), His6-BARD1-(25–189), His6-BRCA1-(1–304), GST-BRCA1-(1–342) and GST-BARD1-(1–111) were produced in BL21/DE3 bacteria by induction with 0.4 mmIPTG for 12 h at 25 °C. Cells were lysed in buffer containing 50 mm Tris-HCl, pH 8.0, 0.5% NP-40, 1% Triton X-100, 50 mm NaCl, 1 mm DTT, 1 mm EDTA, 10% glycerol, and protease inhibitors, and the proteins were purified either with nickel beads (Qiagen) or glutathione-agarose beads (Sigma) according to the manufacturer's instructions. The procedure for the in vitroUb ligation assay was essentially the same as previously described (7Ohta T. Michel J.J. Schottelius A.J. Xiong Y. Mol. Cell. 1999; 3: 535-541Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar,18Ohta T. Michel J.J. Xiong Y. Oncogene. 1999; 18: 6758-6766Crossref PubMed Scopus (44) Google Scholar). The BRCA1-BARD1 immunocomplexes immobilized on protein A-agarose beads were washed three times with buffer A and two times with buffer B containing 25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.01% Nonidet P-40, 10% glycerol, and 1 mm EDTA, and added to a ubiquitin ligation reaction mixture (30 μl) that contained 50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 2 mm NaF, 10 nm okadaic acid, 2 mmATP, 0.6 mm DTT, 0.75 μg of 32P-Ub, 40 ng of E1, and 0.3 μg of E2 protein. After incubation for 30 min at 37 °C with shaking, the reactions were terminated by boiling in Laemmli SDS-loading buffer with 0.1 m DTT, and half of the sample was resolved by SDS-PAGE followed by autoradiography. For the in vitro Ub ligation assay using the purified RING finger domain of BRCA1 or BARD1, GST-fused proteins bound to glutathione-agarose beads, and eluted His6-tagged proteins were used instead of immunocomplexes. The activity of E2 was analyzed by the same procedure without E3. The discovery that the small RING finger proteins, ROC1 and ROC2, are ubiquitin ligases prompted us to determine whether BRCA1 and BARD1, two of the most important RING finger proteins implicated in breast cancer, also function as ubiquitin ligases. We first determined which E2/Ubc could be activated by BRCA1 or BARD1. Five representative mammalian E2s (UbcH1, UbcH2, Cdc34, UbcH5c and UbcH7) were purified from bacteria (Fig.1 A). Individual E2s were incubated with E1 and 32P-Ub in the presence of ATP to verify their Ub binding capacity. Four of the five E2s (UbcH2, Cdc34, UbcH5c, and UbcH7) were determined to be active enzymes by judging the ability to bind to 32P-Ub (Fig. 1 B, lanes 3–6, lower panel), and was dissociated by addition of DTT (lanes 3–6, upper panel). When the anti-Myc immunocomplex derived from Myc-BRCA1-(1–772) or Myc-BARD1-transfected 293T cells was added to the reaction, only UbcH5c among the five E2s was capable of promoting ubiquitin polymerization with the immunocomplex as determined by the appearance of a high molecular weight 32P smear (Fig. 1 C, lanes 4 and 9). Although a crystal structure study revealed the interaction between the RING finger of c-Cbl and UbcH7 (19Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar), UbcH7 was deficient in collaborating with the RING finger of BRCA1 or BARD1 to promote Ub polymerization (lanes 5 and 10). These results suggest that the BRCA1- and BARD1-associated protein complexes contain Ub ligase activities. We noted that the Ub ligase activities of the BRCA1 and BARD1 immunocomplexes, which were only detectable after long exposure of the film (Fig. 1 C), were very low and sought conditions for higher activity. Because it was reported that BRCA1 and BARD1 proteins preferentially formed stable heterodimers (6Meza J.E. Brzovic P.S. King M-C. Klevit R.E. J. Biol. Chem. 1999; 274: 5659-5665Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), we next tested whether the BRCA1-BARD1 heterodimer complex formation affects Ub ligase activity. Surprisingly, when Myc-BRCA1-(1–772) was co-transfected with HA-BARD1, the anti-Myc immunocomplex exhibited significantly higher Ub ligase activity (Fig.2 A, lane 5) than that of either the Myc-BARD1 (lane 3) or Myc-BRCA1-(1–772) (lane 4) single transfection. It is possible that the low Ub ligase activity detected with the immunocomplexes from single transfections of BRCA1 or BARD1 be by with the RING finger protein. addition of Myc-BRCA1-(1–772) dramatically the Ub ligase activity of the immunocomplex (lane E2F1 and cyclin the proteins to interact with the N terminus of BRCA1 H. N. J. Oncogene. PubMed Scopus Google Scholar), as as a protein that to BARD1 Science. 1999; PubMed Scopus Google Scholar) not have a on Ub ligase activity of BRCA1 or BARD1 (Fig. 2 the that the with BRCA1-BARD1 is mediated by the structure of each protein by its and verified the between these proteins (lane verify the of the RING finger in activity and to the relationship between the BRCA1 function and the Ub ligase activity, we constructs containing BRCA1 with a mutation at P.S. J. King M.C. Klevit R.E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The mutation completely Ub ligase activity (Fig. 2 C, lane 3) when with the anti-Myc immunocomplex precipitated from cells with Myc-BRCA1-(1–772) and (lane and the activity of the immunocomplex to the level of the single transfection (lanes mutation in the RING finger also disrupted the activity to the same as (lanes 4 3 and the and the RING mutations to of and not the Ub ligase activity, a detectable of the activity was (Fig. 2 lanes 3 and the that the ligase activity in the immunocomplex is by proteins and also to determine the of the Ub ligase activity, the RING finger domain of BRCA1 and of BARD1 were purified from bacteria via or A). with the a mixture of the purified RING finger domains of BRCA1 and BARD1 exhibited significant Ub ligase activity (Fig. 3 B, lanes and either the BRCA1 (lanes 1 and 5) or BARD1 (lanes 2 and RING detectable Ub ligase activity. His6-E2F1 not the Ub ligase activity of BRCA1 (lane it interact with GST-BRCA1-(1–342) not RING finger domains comprising residues 1–304 of BRCA1 and residues 25–189 of BARD1 were capable of polymerizing ubiquitin (lane BRCA1C61G, the breast cancer-derived RING finger mutant, not with BARD1 to UbcH5c (lane these results that BARD1 and BRCA1 Ub ligase activity with the E2 The contained at and (Fig. the same as that in the assay (Fig. These are to be ubiquitin or ubiquitin bound to small such as The of the these not from the BRCA1-BARD1 complex when washed with buffer B not is a that these two molecules are ubiquitination substrates of each for We analyzed the steady-state levels of these proteins by Polyubiquitinated substrates are rapidly degraded by the 26 S proteasome the steady-state level of such substrates to However, the steady-state level of Myc-BRCA1-(1–772) was increased after co-transfection of in a A). the steady-state level of was increased when it was with Myc-BRCA1-(1–772) (Fig. These results that BRCA1 and BARD1 each the ability to the polyubiquitin suggesting that are not substrates by each for The RING finger motif was to be a DNA binding site when BRCA1 was first identified as a for breast and ovarian cancer, it was to the a DNA binding motif (1Zheng L. Li S. Boyer T.G. Lee W.H. Oncogene. 2000; 19: 6159-6175Crossref PubMed Scopus (136) Google Scholar). the of the domain in that BRCA1 is a and the RING finger in DNA However, binding between the RING finger of BRCA1 and DNA has not been the RING finger of BRCA1 has been to be important for a interaction with RING finger which was identified and the BARD1 (5Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.C. Hwang L.Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (626) Google Scholar). A biochemical that the interaction between BRCA1 and BARD1 was mediated by regions the RING finger motif (6Meza J.E. Brzovic P.S. King M-C. Klevit R.E. J. Biol. Chem. 1999; 274: 5659-5665Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), suggesting that the two RING fingers contained function the of the two was In the report, we evidence demonstrating that the RING heterodimer BRCA1-BARD1 functions as a Ub the BRCA1-BARD1 immunocomplex from BRCA1- and cells contains a significant ability to promote vitro, and of either of the two from the transfection this activity. the purified RING finger domains of BRCA1 and BARD1 exhibit high Ub ligase activity. of either of the two RING fingers this activity. in a breast cancer-derived RING finger mutation of BRCA1, the ability to promote Although it is possible that the major reaction in the study is BRCA1 and BARD1 are not substrates by each for each in A of RING finger ubiquitin ligases is R. Yasuda H. Oncogene. 2000; 19: 1473-1476Crossref PubMed Scopus (315) Google Scholar, 14Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (916) Google Scholar, 15Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (873) Google Scholar, 16Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (947) Google and T. Michel J.J. Xiong Y. Oncogene. 1999; 18: 6758-6766Crossref PubMed Scopus (44) Google Scholar). The of in the function of RING ubiquitin ligases to be BRCA1-BARD1 a of RING containing Ub ligase that a heterodimeric RING finger However, the of RING finger molecules in complexes of RING Ub ligases has not been It to be determined whether either as or between two RING fingers a general for activating Ub ligase activity. this can be detected in immunocomplexes precipitated from cells transfected with and the S of the cell the steady-state levels of BRCA1 a and BARD1 with BRCA1 into Y. Xu X.L. Yang M.C. Bowcock A.M. Baer R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). it is that an in vivo Ub ligase activity of BRCA1-BARD1 a in S the formation of the heterodimer is a in the of BRCA1 and in vivo Ub ligase activity to be the function of the BRCA1-BARD1 Ub the substrates for is proteins that are degraded or in S be substrates for the BRCA1-BARD1 Ub The high Ub ligase activity of BRCA1-BARD1 The that the two RING finger domains of BRCA1 and BARD1 are for biochemical function that the or the by mutations in the domains of two molecules be the mutations in in breast and ovarian cancers have been reported Tsan J.T. Y. Phung A. Spillman M.A. R. J.M. Baer R. Bowcock A.M. Mol. Genet. 1998; PubMed Scopus Google Scholar). BARD1 is potentially as important a as BRCA1 in of breast and ovarian each mutation of the two molecules and vitro Ub ligase activity, it be possible to the for breast and ovarian cancer in with point mutations in the RING finger domains of BRCA1 or BARD1. the that the BRCA1-BARD1 heterodimer complex as a Ub ligase a to into the of BRCA1 and BARD1 in We Dr. Yue Xiong and W. for and of the Dr. Lee for BRCA1 We also for