title: Autoubiquitination of the BRCA1*BARD1 RING ubiquitin ligase.
abstract: The RING finger of BRCA1 confers ubiquitin ligase activity that is markedly enhanced when complexed with another RING-containing protein, BARD1, and is required for the function of this tumor suppressor protein in protecting genomic integrity. Here, we report that co-expression of BRCA1-(1-639) and BARD1 in bacteria can assemble a potent ubiquitin ligase activity. Purified BRCA1-(1-639)*BARD1 stimulated the Ubc5c-mediated monoubiquitination of histone H2A/H2AX in vitro, suggesting a possible role for BRCA1*BARD1 in modifying chromatin structure. Moreover, the truncated BRCA1*BARD1 complex exhibited efficient autoubiquitination activity in vitro capable of assembling non-lysine 48-linked polyubiquitin chains on both BRCA1-(1-639) and BARD1. When co-expressed in cells by transient transfection, the recombinant BRCA1-(1-300).BARD1 complex was found to be associated with polyubiquitin chains, suggesting that BRCA1-(1-300)*BARD1 was ubiquitinated in vivo as well. These results raise the possibility that BRCA1*BARD1 acts to assemble non-lysine 48-linked polyubiquitin chains that may serve as part of a signaling platform required for coordinating DNA repair-related events.
authors.affiliation: Derald H. Ruttenberg Cancer Center, The Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
authors.forename: Angus
authors.initials: A
authors.lastname: Chen
authors.forename: Frida E
authors.initials: FE
authors.lastname: Kleiman
authors.forename: James L
authors.initials: JL
authors.lastname: Manley
authors.forename: Toru
authors.initials: T
authors.lastname: Ouchi
authors.forename: Zhen-Qiang
authors.initials: ZQ
authors.lastname: Pan
mesh_terms: D019313:BRCA1 Protein / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D004260:DNA Repair; D019281:Dimerization; D005982:Glutathione Transferase / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
chemical_list: D019313:BRCA1 Protein; D002352:Carrier Proteins; D006657:Histones; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D008239:Lysine