Among the three Saccharomyces cerevisiae DNA repair epistasis groups, the RAD6 group is the most complicated and least characterized, primarily because it consists of two separate repair pathways: an error-free postreplication repair pathway, and a mutagenesis pathway. The rad6 and rad18 mutants are defective in both pathways, and the rev3 mutant affects only the mutagenesis pathway, but a yeast gene that is involved only in error-free postreplication repair has not been reported. We cloned the MMS2 gene from a yeast genomic library by functional complementation of the mms2-1 mutant [Prakash, L. & Prakash, S. (1977) Genetics 86, 33–55]. MMS2 encodes a 137-amino acid, 15.2-kDa protein with significant sequence homology to a conserved family of ubiquitin-conjugating (Ubc) proteins. However, Mms2 does not appear to possess Ubc activity. Genetic analyses indicate that the mms2 mutation is hypostatic to rad6 and rad18 but is synergistic with the rev3 mutation, and the mms2 mutant is proficient in UV-induced mutagenesis. These phenotypes are reminiscent of a pol30-46 mutant known to be impaired in postreplication repair. The mms2 mutant also displayed a REV3 -dependent mutator phenotype, strongly suggesting that the MMS2 gene functions in the error-free postreplication repair pathway, parallel to the REV3 mutagenesis pathway. Furthermore, with respect to UV sensitivity, mms2 was found to be hypostatic to the rad6 _Δ1–9 mutation, which results in the absence of the first nine amino acids of Rad6. On the basis of these collective results, we propose that the mms2 null mutation and two other allele-specific mutations, rad6 _Δ1–9 and pol30-46, define the error-free mode of DNA postreplication repair, and that these mutations may enhance both spontaneous and DNA damage-induced mutagenesis.

Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13–Mms2 is required for DNA damage repair but not nuclear factor κB (NF-κB) activation, whereas Ubc13–Uev1A is involved in NF-κB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

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. TheMMS2 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 (¹H¹⁵N 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.