Noncovalent Interaction between Ubiquitin and the Human DNA Repair Protein Mms2 Is Required for Ubc13-mediated Polyubiquitination

Abstract

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 1 H 15 N HSQC ( 1 H 15 N 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.

Identification of a protein essential for a major pathway used by human cells to avoid UV- induced DNA damage

“…Likewise, Xiao et al (32) proposed that in yeast, the Mms2͞Ubc13 complex is required to promote both RAD5-and POL30-dependent error-free replication pathways. Recently, McKenna et al (33) showed that in human cells, hMms2 forms a complex with hUbc13, and this interaction is required for hUbc13-mediated polyubiquitination through Lys-63. The crystal structure of this complex also was determined recently (34).…”

Molecular identification and interaction assay of the gene (OsUbc13) encoding a ubiquitin-conjugating enzyme in rice

“…We demonstrated here that OsUbc13 physically interacted with OsCROC-1 ( Fig. 5b; Table 1), which is essential for Ubc13-mediated Lys63 polyubiquitylation (Hofmann and Pickart, 1999;McKenna et al, 2001;Wen et al, 2006); this once again implies the existence of error-free DDT in plants and the critical role of OsUbc13 in tolerance to DNA damage during seed and pollen germination.…”

“…Conventional poly-Ub via Gly76-Lys48 is the principal signal for proteolysis through 26S proteasomes (Hochstrasser, 1996), while Gly76-Lys63 poly-Ub regulates diverse activities in a non-proteolytic way (Pickart, 2001). So far, Ubc13 is the only known Ubc capable of catalyzing the Lys63-linked polyubiquitylation reaction and this function requires interaction with the Ubc variant (Uev, which shares a similar sequence and structure with the E2 Ub-conjugating enzymes, but lacks the active cysteine residue); thus, it is unlike other Ubc proteins that catalyze a conventional Lys48-linked polyubiquitylation reaction (Hofmann and Pickart, 1999;McKenna et al, 2001).…”

RAD5a and REV3 function in two alternative pathways of DNA-damage tolerance in Arabidopsis

“…This observation is understandable, as mammalian Ubc13 interacts with a panel of E3 ligases and is involved in several biological processes in addition to DNA damage responses (Andersen et al, 2005). Since a Uev is absolutely required for Ubc13-mediated Lys63-linked polyubiquitination (Hofmann and Pickart, 1999;McKenna et al, 2001), we suspect that the four AtUEV1 genes may also have distinct as well as overlapping functions, making them unsuitable for the proposed investigation. We turned our attention to plant homolog(s) of Rad5, the yeast cognate E3 ligase of Ubc13 required for error-free DDT (Ulrich and Jentsch, 2000;Xiao et al, 2000), since E3s often provide substrate specificity.…”

“…In contrast to the function in proteasomal protein degradation of target proteins by the K48-linked polyubiquitination, K63-linked polyubiquitination plays diverse non-proteolytic functions (Pickart, 2001). So far, Ubc13 is the only known E2 enzyme capable of catalyzing K63-linked polyubiquitination, which requires an Ubc/E2 variant (Uev) as a co-factor (Hofmann and Pickart, 2001;McKenna et al, 2001). Uev proteins are similar to Ubcs, but lack of a critical Cys residue in the active site.…”