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Reference: Carlile CC, et al. (2009) Synthesis of free and proliferating cell nuclear antigen-bound polyubiquitin chains by the RING E3 ubiquitin ligase Rad5. J Biol Chem 284(43):29326-34

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Abstract

In replicating yeast, lysine 63 (K63)-linked polyubiquitin (polyUb) chains are extended from the ubiquitin (Ub) moiety of monoubiquitinated-PCNA (monoUb-PCNA) by the E2-E3 complex of (Ubc13-Mms2)-Rad5. This promotes error-free bypass of DNA damage lesions. The unusual ability of Ubc13-Mms2 to synthesize unanchored K63-linked polyUb chains in vitro allowed us to resolve the individual roles that it and Rad5 play in the catalysis and specificity of PCNA polyubiquitination. We found that Rad5 stimulates the synthesis of free polyUb chains by Ubc13-Mms2 by enhancing the reactivity of the Ubc13~Ub thiolester bond. Polyubiquitination of monoUb-PCNA was further enhanced by interactions between the N-terminal domain of Rad5 and PCNA. Thus, Rad5 acts both to align monoUb-PCNA with Ub-charged Ubc13 and to stimulate Ub transfer onto K63 of a Ub acceptor. We also found that Rad5 interacts with PCNA independently of the number of monoubiquitinated subunits in the trimer and that it binds to both unmodified and monoUb-PCNA with similar affinities. These findings indicate that Rad5-mediated recognition of monoUb-PCNA in vivo is likely to depend upon interactions with additional factors at stalled replication forks.

Reference Type
Journal Article
Authors
Carlile CC, Pickart CM, Matunis MJ, Cohen RE
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