Noncovalent interaction between ubiquitin and the human DNA repair proteinmms2 is required for ubc13-mediated polyubiquitination

Ubiquitin-conjugating enzyme variants share significant sequence similarity with typical E2 (ubiquitin-conjugating) enzymes of the protein ubiquitinat ion pathway but lack their characteristic active site cysteine residue. The MMS2 gene of Saccharomyces cerevisiae encodes one such ubiquitin-conjugati ng enzyme variant that is involved in the error-free DNA postreplicative re pair pathway through its association with Ubc13, an E2. The Mms2-Ubc13 hete rodimer is capable of linking ubiquitin molecules to one another through an isopeptide bond between the C terminus and Lys-63. Using highly purified c omponents, we show here that the human forms of Mms2 and Ubc13 associate in to a heterodimer that is stable over a range of conditions. The ubiquitin-t hiol ester form of the heterodimer can be produced by the direct activation of its Ubc13 subunit with El (ubiquitin-activating enzyme) or by the assoc iation of Mms2 with the Ubc13-ubiquitin thiol ester. The activated heterodi mer is capable of transferring its covalently bound ubiquitin to Lys-63 of an untethered ubiquitin molecule, resulting in diubiquitin as the predomina nt species. In H-1 N-15 HSQC (H-1 N-15 heteronuclear single quantum coheren ce) NMR experiments, we have mapped the surface determinants of tethered an d untethered ubiquitin that interact with Mms2 and Ubc13 in both their mono meric and dimeric forms. These results have identified a surface of untethe red ubiquitin that interacts with Mms2 in the monomeric and heterodimeric f orm. Furthermore, the C-terminal tail of ubiquitin does not participate in this interaction. These results suggest that the role of Mms2 is to correct ly 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.