The BRCA1/BARD1 Heterodimer Assembles Polyubiquitin Chains through an Unconventional Linkage Involving Lysine Residue K6 of Ubiquitin

Abstract

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 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 (1Scully R. Livingston D.M. Nature. 2000; 408: 429-432Crossref PubMed Scopus (553) Google Scholar). Its protein product contains an NH2-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 (2Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.-C.W. Hwang L.-Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (615) Google Scholar). The BRCA1/BARD1 heterodimer is stabilized by a 4-helix bundle formed by α-helices that immediately flank the RING domains of both polypeptides (3Brzovic P.S. Rajagopal P. Hoyt D.W. King M.-C. Klevit R.E. Nat. Struct. Biol. PubMed Scopus Google Scholar). cellular BRCA1 polypeptides are found in with BARD1 Baer R. Biol. 2000; PubMed Scopus Google Google the BRCA1/BARD1 heterodimer is to an of BRCA1 R. Genet. PubMed Scopus Google Scholar). is by of a that is that of J.T. Baer R. Biol. PubMed Scopus Google an enzymatic for the BRCA1/BARD1 heterodimer in protein A.M. A.M. A. PubMed Scopus Google A. PubMed Scopus Google R. Biol. PubMed Scopus Google A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google P.S. Klevit R. A. PubMed Scopus Google A. R.E. A. PubMed Scopus Google Scholar). occurs through a involving ubiquitin ubiquitin ubiquitin. a and a ubiquitin ligase PubMed Scopus Google A.M. Nat. Biol. PubMed Scopus Google Scholar). In the of a ubiquitin is to a lysine residue the The can serve as a for of a polyubiquitin chain in target the for of E3 is the of a RING and that the RING domain of BRCA1 can the formation of polyubiquitin chains A.M. A.M. A. PubMed Scopus Google A. PubMed Scopus Google Scholar). the E3 ligase of BRCA1 is by BARD1, and is by the a BRCA1 with R. Biol. PubMed Scopus Google A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google P.S. Klevit R. A. PubMed Scopus Google Scholar). of the cellular functions to its ability to tumor may through the enzymatic of the BRCA1/BARD1 of tumor the natural enzymatic substrates of BRCA1/BARD1 been and the of the functions of substrates are In BRCA1/BARD1 as a that polyubiquitin chains R. Biol. PubMed Scopus Google A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google P.S. Klevit R. A. PubMed Scopus Google Scholar). has seven lysine any of can serve as a of chain PubMed Scopus Google A.M. Nat. Biol. PubMed Scopus Google Scholar). proteins are to K48-linked chains in ubiquitin forms an isopeptide bond with lysine residue the A. A. PubMed Scopus Google Scholar). proteins are for degradation, the isopeptide linkage Biol. PubMed Google Scholar). In chains through K63 been in vivo, and chains at and can in Biol. 14: PubMed Scopus Google Biol. PubMed Google Biol. 1996; PubMed Scopus Google Scholar). and chains target proteins for proteasomal chains non-proteolytic that control various DNA and 2000; PubMed Scopus Google Scholar). we mutation to the linkage specificity of BRCA1/BARD1-dependent BRCA1/BARD1 catalyzes ubiquitin polymerization primarily through lysine residue to form an unconventional of polyubiquitin for of and BRCA1 by full-length BRCA1 with an NH2-terminal and a COOH-terminal the The for of the protein by and full-length BARD1 for of E6-AP with an NH2-terminal by full-length E6-AP a of the for by full-length To a the for a ubiquitin a by PubMed Scopus Google and a of the been To the of ubiquitin of the seven lysine of the ubiquitin to an by of to for ubiquitin and for the of ubiquitin of BRCA1/BARD1 the with the the by with the of the BRCA1/BARD1 of with BRCA1 and BARD1 at a of of for and the cell in with the the cell by at for at and the through a To the BRCA1/BARD1 the with for at The with with a and with and The to and and with for at the with and with The a with and the protein and in at The of BRCA1/BARD1 heterodimer by the of of the BRCA1 with that of known of the of and polypeptides in and purified cell by and in and purified to by a of a Biol. 1996; PubMed Scopus Google Scholar). in and at and by by and the to with proteins by and the to with the ubiquitin through a and a with at The with of the and the ubiquitin with at the protein the ubiquitin and a with and in at by and the of by with that of known of and ubiquitin at for in a and with of ubiquitin of and a of the purified BRCA1/BARD1 heterodimer an of the BRCA1 and with the ubiquitin and and the BRCA1 autoubiquitination and ubiquitin. The with of of and of ubiquitin of of and of ubiquitin the with of the by and by The and and linkage of BRCA1/BARD1 and E6-AP by in with the of ubiquitin In with ubiquitin and with of the seven ubiquitin the BRCA1/BARD1 heterodimer as an E3 ligase and as an with E6-AP as an E3 ligase and as an The of the are in linkage of BRCA1/BARD1 and E6-AP by in with the of ubiquitin In with ubiquitin and with of the seven ubiquitin the BRCA1/BARD1 heterodimer as an E3 ligase and as an with E6-AP as an E3 ligase and as an BRCA1 contains primarily K6-linked chains. BRCA1/BARD1-dependent by and the BRCA1 polypeptides by with a in the of ubiquitin and in the of ubiquitin ubiquitin ubiquitin ubiquitin The of and forms of BRCA1 are the full-length BRCA1/BARD1 with a full-length BRCA1 with an and a COOH-terminal and a to full-length The BRCA1/BARD1 heterodimer purified by a and In a heterodimer the mutation of BRCA1 and purified for ability to ubiquitin polymerization in the of of related and known to A.M. A.M. A. PubMed Scopus Google A. PubMed Scopus Google R. Biol. PubMed Scopus Google A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google P.S. Klevit R. A. PubMed Scopus Google Scholar). The by and the formation of polyubiquitin by with in an by the not the BRCA1/BARD1 a of ubiquitin in seven been with the for the conjugation for in with that to the of and of at a is with of BRCA1 autoubiquitination A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google and that the with ubiquitin polyubiquitin the chain specificity of BRCA1/BARD1-dependent polyubiquitination, we a of ubiquitin in for of the seven lysine and in lysine has been to in polyubiquitination in with that BRCA1/BARD1 catalyzes the formation primarily of K6-linked ubiquitin polymers. with a Biol. PubMed Scopus Google the of polyubiquitination in and that BRCA1/BARD1 may to a formation of and chains. a we the linkage specificity of E6-AP, a ubiquitin ligase that can induce and of the tumor suppressor PubMed Scopus Google can in with the of A. PubMed Scopus Google Scholar). in E6-AP polyubiquitination in the that E6-AP directs the synthesis of K48-linked the linkage of BRCA1/BARD1 and E6-AP, in a of ubiquitin in of the seven lysine residues are with and in the are to with the of residue K6. in polyubiquitination by BRCA1/BARD1 in of the of polyubiquitination in with and not that while BRCA1/BARD1 catalyzes the formation of K6-linked it has to induce synthesis of chains with in formation by E6-AP, that K48-linked chains are the of heterologous substrates of polyubiquitination not been that the BRCA1 of the heterodimer autoubiquitination in A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google Scholar). To determine K6-linked chains are to BRCA1 BRCA1/BARD1-dependent and the resulting of BRCA1 by with the in in the of ubiquitin and In the of of BRCA1 with of BRCA1 autoubiquitination A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google Scholar). In contrast, however, BRCA1 not in with that autoubiquitination primarily BRCA1 with K6-linked chains. The ability of BRCA1/BARD1 to induce K6-linked BRCA1 by the that BRCA1 polyubiquitination occurs in with the ubiquitin that a lysine at residue not with the ubiquitin of an unconventional for ubiquitin as the linkage by BRCA1/BARD1 to the of linkage formation in the with a full-length heterodimer that contains the of both BRCA1 and the ubiquitin and and purified the of that linkage to of lysine for in the ubiquitin a known of ubiquitin synthesis in R.E. PubMed Scopus Google by to a that is not to and ubiquitin in a with that the and the that primarily K48-linked that polyubiquitination through the for can occur in that the E3 is Although the linkage specificity of E6-AP not been the synthesis of chains is with the known ability of E6-AP to induce proteasomal of substrates such as A. PubMed Scopus Google and K48-linked chains can target substrates for proteasomal degradation, K63 chains are to serve non-proteolytic functions 2000; PubMed Scopus Google Scholar). In contrast, however, the cellular functions of polyubiquitin chains are not To the of chains has been in by the of Biol. 1996; PubMed Scopus Google Scholar). forms in it chains in the of an E3 Biol. 1996; PubMed Scopus Google Scholar). suggests that the chain linkage of can by the of the and E3 similar is in that primarily chains in the of E6-AP and chains in the of a of ubiquitin and that BRCA1/BARD1 can and Biol. PubMed Scopus Google Scholar). are with the of a comprehensive of ubiquitin that is the linkage specificity of BRCA1/BARD1 to a the formation of K63 BRCA1/BARD1-dependent the functions of K6-linked not been the functions of BRCA1 the natural substrates of BRCA1/BARD1-dependent are The polyubiquitination to is BRCA1 autoubiquitination in A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google Scholar). we that autoubiquitination in conjugation of chains to In of it may that the BRCA1 and BARD1 polypeptides to in J.T. Baer R. Biol. PubMed Scopus Google R. Biol. PubMed Scopus Google Livingston D.M. A. PubMed Scopus Google Scholar). BRCA1 autoubiquitination is by the BRCA1/BARD1 heterodimer by BRCA1 A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google it that it occurs in vivo, to target BRCA1 for proteasomal in suggests that K6-linked chains may not to determine the functions of K6-linked polyubiquitin and its in tumor The BRCA1 tumor suppressor gene has been implicated in various cellular processes that include DNA repair, transcriptional regulation, and cell cycle checkpoint control (1Scully R. Livingston D.M. Nature. 2000; 408: 429-432Crossref PubMed Scopus (553) Google Scholar). Its protein product contains an NH2-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 (2Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.-C.W. Hwang L.-Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (615) Google Scholar). The BRCA1/BARD1 heterodimer is stabilized by a 4-helix bundle formed by α-helices that immediately flank the RING domains of both polypeptides (3Brzovic P.S. Rajagopal P. Hoyt D.W. King M.-C. Klevit R.E. Nat. Struct. Biol. PubMed Scopus Google Scholar). cellular BRCA1 polypeptides are found in with BARD1 Baer R. Biol. 2000; PubMed Scopus Google Google the BRCA1/BARD1 heterodimer is to an of BRCA1 R. Genet. PubMed Scopus Google Scholar). is by of a that is that of J.T. Baer R. Biol. PubMed Scopus Google Scholar). an enzymatic for the BRCA1/BARD1 heterodimer in protein A.M. A.M. A. PubMed Scopus Google A. PubMed Scopus Google R. Biol. PubMed Scopus Google A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google P.S. Klevit R. A. PubMed Scopus Google A. R.E. A. PubMed Scopus Google Scholar). occurs through a involving ubiquitin ubiquitin ubiquitin. a and a ubiquitin ligase PubMed Scopus Google A.M. Nat. Biol. PubMed Scopus Google Scholar). In the of a ubiquitin is to a lysine residue the The can serve as a for of a polyubiquitin chain in target the for of E3 is the of a RING and that the RING domain of BRCA1 can the formation of polyubiquitin chains A.M. A.M. A. PubMed Scopus Google A. PubMed Scopus Google Scholar). the E3 ligase of BRCA1 is by BARD1, and is by the a BRCA1 with R. Biol. PubMed Scopus Google A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google P.S. Klevit R. A. PubMed Scopus Google Scholar). of the cellular functions to its ability to tumor may through the enzymatic of the BRCA1/BARD1 of tumor the natural enzymatic substrates of BRCA1/BARD1 been and the of the functions of substrates are In BRCA1/BARD1 as a that polyubiquitin chains R. Biol. PubMed Scopus Google A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google P.S. Klevit R. A. PubMed Scopus Google Scholar). has seven lysine any of can serve as a of chain PubMed Scopus Google A.M. Nat. Biol. PubMed Scopus Google Scholar). proteins are to K48-linked chains in ubiquitin forms an isopeptide bond with lysine residue the A. A. PubMed Scopus Google Scholar). proteins are for degradation, the isopeptide linkage Biol. PubMed Google Scholar). In chains through K63 been in vivo, and chains at and can in Biol. 14: PubMed Scopus Google Biol. PubMed Google Biol. 1996; PubMed Scopus Google Scholar). and chains target proteins for proteasomal chains non-proteolytic that control various DNA and 2000; PubMed Scopus Google Scholar). we mutation to the linkage specificity of BRCA1/BARD1-dependent BRCA1/BARD1 catalyzes ubiquitin polymerization primarily through lysine residue to form an unconventional of polyubiquitin for of and BRCA1 by full-length BRCA1 with an NH2-terminal and a COOH-terminal the The for of the protein by and full-length BARD1 for of E6-AP with an NH2-terminal by full-length E6-AP a of the for by full-length To a the for a ubiquitin a by PubMed Scopus Google and a of the been To the of ubiquitin of the seven lysine of the ubiquitin to an by of to for ubiquitin and for the of ubiquitin of BRCA1/BARD1 the with the the by with the of the BRCA1/BARD1 of with BRCA1 and BARD1 at a of of for and the cell in with the the cell by at for at and the through a To the BRCA1/BARD1 the with for at The with with a and with and The to and and with for at the with and with The a with and the protein and in at The of BRCA1/BARD1 heterodimer by the of of the BRCA1 with that of known of the of and polypeptides in and purified cell by and in and purified to by a of a Biol. 1996; PubMed Scopus Google Scholar). in and at and by by and the to with proteins by and the to with the ubiquitin through a and a with at The with of the and the ubiquitin with at the protein the ubiquitin and a with and in at by and the of by with that of known of and ubiquitin at for in a and with of ubiquitin of and a of the purified BRCA1/BARD1 heterodimer an of the BRCA1 and with the ubiquitin and and the BRCA1 autoubiquitination and ubiquitin. The with of of and of ubiquitin of of and of ubiquitin the with of the by and by The and and linkage of BRCA1/BARD1 and E6-AP by in with the of ubiquitin In with ubiquitin and with of the seven ubiquitin the BRCA1/BARD1 heterodimer as an E3 ligase and as an with E6-AP as an E3 ligase and as an BRCA1 contains primarily K6-linked chains. BRCA1/BARD1-dependent by and the BRCA1 polypeptides by with a in the of ubiquitin and in the of ubiquitin ubiquitin ubiquitin ubiquitin The of and forms of BRCA1 are the for of and BRCA1 by full-length BRCA1 with an NH2-terminal and a COOH-terminal the The for of the protein by and full-length BARD1 for of E6-AP with an NH2-terminal by full-length E6-AP a of the for by full-length To a the for a ubiquitin a by PubMed Scopus Google and a of the been To the of ubiquitin of the seven lysine of the ubiquitin to an by of to for ubiquitin and for the of ubiquitin of BRCA1/BARD1 the with the the by with the of the BRCA1/BARD1 of with BRCA1 and BARD1 at a of of for and the cell in with the the cell by at for at and the through a To the BRCA1/BARD1 the with for at The with with a and with and The to and and with for at the with and with The a with and the protein and in at The of BRCA1/BARD1 heterodimer by the of of the BRCA1 with that of known of the of and polypeptides in and purified cell by and in and purified to by a of a Biol. 1996; PubMed Scopus Google Scholar). in and at and by by and the to with proteins by and the to with the ubiquitin through a and a with at The with of the and the ubiquitin with at the protein the ubiquitin and a with and in at by and the of by with that of known of and ubiquitin In at for in a and with of ubiquitin of and a of the purified BRCA1/BARD1 heterodimer an of the BRCA1 and with the ubiquitin and and the BRCA1 autoubiquitination and ubiquitin. The with of of and of ubiquitin of of and of ubiquitin the with of the by and by The and and full-length BRCA1/BARD1 with a full-length BRCA1 with an and a COOH-terminal and a to full-length The BRCA1/BARD1 heterodimer purified by a and In a heterodimer the mutation of BRCA1 and purified for ability to ubiquitin polymerization in the of of related and known to A.M. A.M. A. PubMed Scopus Google A. PubMed Scopus Google R. Biol. PubMed Scopus Google A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google P.S. Klevit R. A. PubMed Scopus Google Scholar). The by and the formation of polyubiquitin by with in an by the not the BRCA1/BARD1 a of ubiquitin in seven been with the for the conjugation for in with that to the of and of at a is with of BRCA1 autoubiquitination A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google and that the with ubiquitin polyubiquitin the chain specificity of BRCA1/BARD1-dependent polyubiquitination, we a of ubiquitin in for of the seven lysine and in lysine has been to in polyubiquitination in with that BRCA1/BARD1 catalyzes the formation primarily of K6-linked ubiquitin polymers. with a Biol. PubMed Scopus Google the of polyubiquitination in and that BRCA1/BARD1 may to a formation of and chains. a we the linkage specificity of E6-AP, a ubiquitin ligase that can induce and of the tumor suppressor PubMed Scopus Google can in with the of A. PubMed Scopus Google Scholar). in E6-AP polyubiquitination in the that E6-AP directs the synthesis of K48-linked the linkage of BRCA1/BARD1 and E6-AP, in a of ubiquitin in of the seven lysine residues are with and in the are to with the of residue K6. in polyubiquitination by BRCA1/BARD1 in of the of polyubiquitination in with and not that while BRCA1/BARD1 catalyzes the formation of K6-linked it has to induce synthesis of chains with in formation by E6-AP, that K48-linked chains are the of heterologous substrates of polyubiquitination not been that the BRCA1 of the heterodimer autoubiquitination in A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google Scholar). To determine K6-linked chains are to BRCA1 BRCA1/BARD1-dependent and the resulting of BRCA1 by with the in in the of ubiquitin and In the of of BRCA1 with of BRCA1 autoubiquitination A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google Scholar). In contrast, however, BRCA1 not in with that autoubiquitination primarily BRCA1 with K6-linked chains. The ability of BRCA1/BARD1 to induce K6-linked BRCA1 by the that BRCA1 polyubiquitination occurs in with the ubiquitin that a lysine at residue not with the ubiquitin To full-length BRCA1/BARD1 with a full-length BRCA1 with an and a COOH-terminal and a to full-length The BRCA1/BARD1 heterodimer purified by a and In a heterodimer the mutation of BRCA1 and purified for ability to ubiquitin polymerization in the of of related and known to A.M. A.M. A. PubMed Scopus Google A. PubMed Scopus Google R. Biol. PubMed Scopus Google A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google P.S. Klevit R. A. PubMed Scopus Google Scholar). The by and the formation of polyubiquitin by with in an by the not the BRCA1/BARD1 a of ubiquitin in seven been with the for the conjugation for in with that to the of and of at a is with of BRCA1 autoubiquitination A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google and that the with ubiquitin polyubiquitin chains. To the chain specificity of BRCA1/BARD1-dependent polyubiquitination, we a of ubiquitin in for of the seven lysine and in lysine has been to in polyubiquitination in with that BRCA1/BARD1 catalyzes the formation primarily of K6-linked ubiquitin polymers. with a Biol. PubMed Scopus Google the of polyubiquitination in and that BRCA1/BARD1 may to a formation of and chains. a we the linkage specificity of E6-AP, a ubiquitin ligase that can induce and of the tumor suppressor PubMed Scopus Google can in with the of A. PubMed Scopus Google Scholar). in E6-AP polyubiquitination in the that E6-AP directs the synthesis of K48-linked chains. To the linkage of BRCA1/BARD1 and E6-AP, in a of ubiquitin in of the seven lysine residues are with and in the are to with the of residue K6. in polyubiquitination by BRCA1/BARD1 in of the of polyubiquitination in with and not that while BRCA1/BARD1 catalyzes the formation of K6-linked it has to induce synthesis of chains with in formation by E6-AP, that K48-linked chains are the of Although heterologous substrates of polyubiquitination not been that the BRCA1 of the heterodimer autoubiquitination in A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google Scholar). To determine K6-linked chains are to BRCA1 BRCA1/BARD1-dependent and the resulting of BRCA1 by with the in in the of ubiquitin and In the of of BRCA1 with of BRCA1 autoubiquitination A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google Scholar). In contrast, however, BRCA1 not in with that autoubiquitination primarily BRCA1 with K6-linked chains. The ability of BRCA1/BARD1 to induce K6-linked BRCA1 by the that BRCA1 polyubiquitination occurs in with the ubiquitin that a lysine at residue not with the ubiquitin of an unconventional for ubiquitin as the linkage by BRCA1/BARD1 to the of linkage formation in the with a full-length heterodimer that contains the of both BRCA1 and the ubiquitin and and purified the of that linkage to of lysine for in the ubiquitin a known of ubiquitin synthesis in R.E. PubMed Scopus Google by to a that is not to and ubiquitin in a with that the and the that primarily K48-linked that polyubiquitination through the for can occur in that the E3 is Although the linkage specificity of E6-AP not been the synthesis of chains is with the known ability of E6-AP to induce proteasomal of substrates such as A. PubMed Scopus Google and K48-linked chains can target substrates for proteasomal degradation, K63 chains are to serve non-proteolytic functions 2000; PubMed Scopus Google Scholar). In contrast, however, the cellular functions of polyubiquitin chains are not To the of chains has been in by the of Biol. 1996; PubMed Scopus Google Scholar). forms in it chains in the of an E3 Biol. 1996; PubMed Scopus Google Scholar). suggests that the chain linkage of can by the of the and E3 similar is in that primarily chains in the of E6-AP and chains in the of a of ubiquitin and that BRCA1/BARD1 can and Biol. PubMed Scopus Google Scholar). are with the of a comprehensive of ubiquitin that is the linkage specificity of BRCA1/BARD1 to a the formation of K63 BRCA1/BARD1-dependent the functions of K6-linked not been the functions of BRCA1 the natural substrates of BRCA1/BARD1-dependent are The polyubiquitination to is BRCA1 autoubiquitination in A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google Scholar). we that autoubiquitination in conjugation of chains to In of it may that the BRCA1 and BARD1 polypeptides to in J.T. Baer R. Biol. PubMed Scopus Google R. Biol. PubMed Scopus Google Livingston D.M. A. PubMed Scopus Google Scholar). BRCA1 autoubiquitination is by the BRCA1/BARD1 heterodimer by BRCA1 A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google it that it occurs in vivo, to target BRCA1 for proteasomal in suggests that K6-linked chains may not to determine the functions of K6-linked polyubiquitin and its in tumor The of an unconventional for ubiquitin as the linkage by BRCA1/BARD1 to the of linkage formation in the with a full-length heterodimer that contains the of both BRCA1 and the ubiquitin and and purified the of that linkage to of lysine for in the ubiquitin a known of ubiquitin synthesis in R.E. PubMed Scopus Google by to a that is not to and ubiquitin in a with that the and the that primarily K48-linked that polyubiquitination through the for can occur in that the E3 is Although the linkage specificity of E6-AP not been the synthesis of chains is with the known ability of E6-AP to induce proteasomal of substrates such as A. PubMed Scopus Google Scholar). and K48-linked chains can target substrates for proteasomal degradation, K63 chains are to serve non-proteolytic functions 2000; PubMed Scopus Google Scholar). In contrast, however, the cellular functions of polyubiquitin chains are not To the of chains has been in by the of Biol. 1996; PubMed Scopus Google Scholar). forms in it chains in the of an E3 Biol. 1996; PubMed Scopus Google Scholar). suggests that the chain linkage of can by the of the and E3 similar is in that primarily chains in the of E6-AP and chains in the of a of ubiquitin and that BRCA1/BARD1 can and Biol. PubMed Scopus Google Scholar). are with the of a comprehensive of ubiquitin that is the linkage specificity of BRCA1/BARD1 to a the formation of K63 BRCA1/BARD1-dependent the functions of K6-linked not been the functions of BRCA1 the natural substrates of BRCA1/BARD1-dependent are The polyubiquitination to is BRCA1 autoubiquitination in A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google Scholar). we that autoubiquitination in conjugation of chains to In of it may that the BRCA1 and BARD1 polypeptides to in J.T. Baer R. Biol. PubMed Scopus Google R. Biol. PubMed Scopus Google Livingston D.M. A. PubMed Scopus Google Scholar). BRCA1 autoubiquitination is by the BRCA1/BARD1 heterodimer by BRCA1 A. Biol. PubMed Scopus Google Biol. PubMed Scopus Google PubMed Scopus Google it that it occurs in vivo, to target BRCA1 for proteasomal in suggests that K6-linked chains may not to determine the functions of K6-linked polyubiquitin and its in tumor and for and the are to for ubiquitin