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E2–BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages


An E3 ubiquitin ligase mediates the transfer of activated ubiquitin from an E2 ubiquitin-conjugating enzyme to its substrate lysine residues. Using a structure-based, yeast two-hybrid strategy, we discovered six previously unidentified interactions between the human heterodimeric RING E3 BRCA1-BARD1 and the human E2s UbcH6, Ube2e2, UbcM2, Ubc13, Ube2k and Ube2w. All six E2s bind directly to the BRCA1 RING motif and are active with BRCA1-BARD1 for autoubiquitination in vitro. Four of the E2s direct monoubiquitination of BRCA1. Ubc13-Mms2 and Ube2k direct the synthesis of Lys63- or Lys48-linked ubiquitin chains on BRCA1 and require an acceptor ubiquitin attached to BRCA1. Differences between the mono- and polyubiquitination activities of the BRCA1-interacting E2s correlate with their ability to bind ubiquitin noncovalently at a site distal to the active site. Thus, BRCA1 has the ability to direct the synthesis of specific polyubiquitin chain linkages, depending on the E2 bound to its RING.

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Figure 1: A fused BRCA1-BARD1 construct interacts with multiple E2s in a yeast two-hybrid analysis.
Figure 2: Confirmation of direct E2–BRCA1-BARD1 interactions by NMR.
Figure 3: Sequence alignment of helix 1, loop L1 and loop L2 of BRCA1-interacting and noninteracting E2s.
Figure 4: Mutation of conserved alanine in loop L2 of UbcH5c eliminates autoubiquitination of BRCA1.
Figure 5: Autoubiquitination activity assays of BRCA1 with its interacting E2s.
Figure 6: Model of BRCA1-BARD1 autoubiquitination mechanisms.

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This paper is dedicated to the memory of C.M. Pickart. We thank C. Eakin, M. Daley and D.M. Wenzel for careful reading of the manuscript; C. Parchen and A. Lissounov for assistance with molecular cloning and protein characterization; D.M. Wenzel for sharing unpublished data; and the following researchers for plasmids encoding human E2s: T. Ohta (St. Marianna University; Rad6b, UbcH10 and Ube2k), B. Schulman (St. Jude Children's Research Hospital; UbcH5b and UbcH12), K. Lorick (US National Cancer Institute; Ube2h, UbcH8, Ubc13 and Cdc34), S. Plafker (University of Oklahoma Health Sciences Center; UbcM2), S. Lin (Wesleyan University; Uev1a), and W. Xiao (University of Saskatchewan; Mms2). D.E.C. was supported by a US Department of Defense Breast Cancer Research Program fellowship (W81XWH-04-1-0390) and a US National Institute of General Medical Sciences training grant (T32GM007270). This work was supported by US National Cancer Institute grant R01 CA079953 to R.E.K.

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D.E.C., P.S.B. and R.E.K. designed and analyzed the experiments. D.E.C. conducted all experiments except those in Supplementary Figures 4 and 6, which were conducted by P.S.B. The paper was written by D.E.C. and R.E.K. with editorial assistance from P.S.B.

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Correspondence to Rachel E Klevit.

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Christensen, D., Brzovic, P. & Klevit, R. E2–BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages. Nat Struct Mol Biol 14, 941–948 (2007).

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