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Papers In Press, published online ahead of print August 14, 2001
J. Biol. Chem, 10.1074/jbc.M102858200
Submitted on March 30, 2001
Revised on August 14, 2001
Accepted on August 13, 2001
Biochemistry, University of Alberta, Edmonton, AB T6G 2H7
Corresponding Author: mike.ellison{at}ualberta.ca
Ubiquitin-conjugating enzyme variants share significant sequence similarity with typical E2 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 Lys63. 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-thiolester form of the heterodimer can be produced by the direct activation of its Ubc13 subunit with E1, or by the association of Mms2 with the Ubc13-ubiquitin thiolester. The activated heterodimer is capable of transferring its covalently bound ubiquitin to Lys63 of an untethered ubiquitin molecule, resulting in di-ubiquitin as the predominant species. We also show here that the human and yeast proteins display markedly different in vitro chain building and autoubiquitination properties. In 1H-15N-HSQC 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 Lys63 is placed proximally to the c-terminus of the ubiquitin molecule that is linked to the active site of Ubc13.
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