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Originally published In Press as doi:10.1074/jbc.M102858200 on August 14, 2001

J. Biol. Chem., Vol. 276, Issue 43, 40120-40126, October 26, 2001
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Noncovalent Interaction between Ubiquitin and the Human DNA Repair Protein Mms2 Is Required for Ubc13-mediated Polyubiquitination*

Sean McKennaDagger §, Leo SpyracopoulosDagger , Trevor MoraesDagger , Landon Pastushok||, Christopher PtakDagger , Wei Xiao||**, and Michael J. EllisonDagger Dagger Dagger

From the Dagger  Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada,  Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, and || Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada

Ubiquitin-conjugating enzyme variants share significant sequence similarity with typical E2 (ubiquitin-conjugating) enzymes of the protein ubiquitination pathway but lack their characteristic active site cysteine residue. The MMS2 gene of Saccharomyces cerevisiae encodes one such ubiquitin-conjugating enzyme variant that is involved in the error-free DNA postreplicative repair pathway through its association with Ubc13, an E2. The Mms2-Ubc13 heterodimer is capable of linking ubiquitin molecules to one another through an isopeptide bond between the C terminus and Lys-63. Using highly purified components, we show here that the human forms of Mms2 and Ubc13 associate into a heterodimer that is stable over a range of conditions. The ubiquitin-thiol ester form of the heterodimer can be produced by the direct activation of its Ubc13 subunit with E1 (ubiquitin-activating enzyme) or by the association of Mms2 with the Ubc13-ubiquitin thiol ester. The activated heterodimer is capable of transferring its covalently bound ubiquitin to Lys-63 of an untethered ubiquitin molecule, resulting in diubiquitin as the predominant species. In 1H 15N HSQC (1H 15N heteronuclear single quantum coherence) NMR experiments, we have mapped the surface determinants of tethered and untethered ubiquitin that interact with Mms2 and Ubc13 in both their monomeric and dimeric forms. These results have identified a surface of untethered ubiquitin that interacts with Mms2 in the monomeric and heterodimeric form. Furthermore, the C-terminal tail of ubiquitin does not participate in this interaction. These results suggest that the role of Mms2 is to correctly orient either a target-bound or untethered ubiquitin molecule such that its Lys-63 is placed proximally to the C terminus of the ubiquitin molecule that is linked to the active site of Ubc13.

* This work was supported by a research grant from the National Cancer Institute of Canada and by Grant MT-15076 from the Canadian Institutes of Health Research (to W. X.).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.

§ A Natural Sciences and Engineering Research Council of Canada scholar.

** A research scientist of the National Cancer Institute of Canada. To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E5, Canada. Tel.: 306-966-4308; Fax.: 306-966-4311; E-mail: xioaow@sask.usask.ca.

Dagger Dagger To whom correspondence should be addressed: Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-5839; Fax.: 780-492-0886. E-mail: mike.ellison@ualberta.ca.

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


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