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Originally published In Press as doi:10.1074/jbc.M103378200 on May 21, 2001

J. Biol. Chem., Vol. 276, Issue 30, 27936-27943, July 27, 2001
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In Vitro Assembly and Recognition of Lys-63 Polyubiquitin Chains*

Roseanne M. HofmannDagger and Cecile M. Pickart§

From the Department of Biochemistry and Molecular Biology, School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205

Polyubiquitin chains assembled through lysine 48 (Lys-48) of ubiquitin act as a signal for substrate proteolysis by 26 S proteasomes, whereas chains assembled through Lys-63 play a mechanistically undefined role in post-replicative DNA repair. We showed previously that the products of the UBC13 and MMS2 genes function in error-free post-replicative DNA repair in the yeast Saccharomyces cerevisiae and form a complex that assembles Lys-63-linked polyubiquitin chains in vitro. Here we confirm that the Mms2·Ubc13 complex functions as a high affinity heterodimer in the chain assembly reaction in vitro and report the results of a kinetic characterization of the polyubiquitin chain assembly reaction. To test whether a Lys-63-linked polyubiquitin chain can signal degradation, we conjugated Lys-63-linked tetra-ubiquitin to a model substrate of 26 S proteasomes. Although the noncanonical chain effectively signaled substrate degradation, the results of new genetic epistasis studies agree with previous genetic data in suggesting that the proteolytic activity of proteasomes is not required for error-free post-replicative repair.

* This work was supported by National Institutes of Health (NIH) Grant GM60372 and by a training grant from the NIEHS, NIH.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Rm. 8041, Johns Hopkins University, 615 N. Wolfe St. Baltimore, MD 21205. Tel.: 410-614-4554; Fax: 410-955-2926; E-mail: cpickart@welchlink.welch.jhu.edu.

Dagger Present address: Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, NY 10021.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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