The BRCA1 tumor suppressor forms a heterodimer with the BARD1 protein, and the resulting complex functions as an E3 ubiquitin ligase that catalyzes the synthesis of polyubiquitin chains. In theory, polyubiquitination can occur by isopeptide bond formation at any of the seven lysine residues of ubiquitin. The isopeptide linkage of a polyubiquitin chain is a particularly important determinant of its cellular function, such that K48-linked chains commonly target proteins for proteasomal degradation, while K63 chains serve non-proteolytic roles in various signaling pathways. To determine the isopeptide linkage formed by BRCA1/BARD1-dependent polyubiquitination, we purified a full-length heterodimeric complex and compared its linkage specificity with that of E6-AP, an E3 ligase known to induce proteolysis of its cellular substrates. Using a comprehensive mutation analysis, we found that E6-AP catalyzes the synthesis of K48-linked polyubiquitin chains. In contrast, however, the BRCA1/BARD1 heterodimer directs polymerization of ubiquitin primarily through an unconventional linkage involving lysine residue K6. Although heterologous substrates of BRCA1/BARD1 are not known, BRCA1 autoubiquitination occurs principally by conjugation with K6-linked polymers. The ability of BRCA1/BARD1 to form K6-linked polyubiquitin chains suggests that it may impart unique cellular properties to its natural enzymatic substrates.The BRCA1 tumor suppressor gene has been implicated in various cellular processes that include DNA repair, transcriptional regulation, and cell cycle checkpoint control (1). Its protein product contains an NH 2 -terminal RING domain and two COOH-terminal BRCT repeats. In vivo, BRCA1 exists as a heterodimer with BARD1, a related protein that displays a similar configuration of RING and BRCT motifs (2). The BRCA1/BARD1 heterodimer is stabilized by a 4-helix bundle formed by ␣-helices that immediately flank the RING domains of both polypeptides (3). Since most cellular BRCA1 polypeptides are found in association with BARD1 (4, 5), the BRCA1/ BARD1 heterodimer is likely to be an essential mediator of BRCA1 function (6). This conclusion is strongly supported by analysis of Bard1-null mice, which display a characteristic phenotype that is essentially indistinguishable from that of Brca1-null animals (7).Recent studies have uncovered an enzymatic role for the BRCA1/BARD1 heterodimer in protein ubiquitination (8 -15). Ubiquitination occurs through a sequential process involving ubiquitin-activating enzyme (E1), 1 a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) (16,17). In the final step of this process, a ubiquitin monomer is covalently attached to a lysine residue on the ultimate substrate. The monoubiquitinated substrate can also serve as a nucleus for assembly of a polyubiquitin chain that, in many cases, will target the substrate for proteasome-mediated degradation. A common feature of many E3 ligases is the presence of a RING motif, and recent studies have established that sequences encompassing the RI...

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