The products of the yeast MMS2 and two human homologs (hMMS2 and CROC-1) define a structurally and functionally conserved Ubc-like protein family

Wei Xiao 2 Stanley L. Lin 1 2 Stacey Broomfield 2 Barbara L. Chow 2 Ying-Fei Wei 0 2 0 Human Genome Sciences Inc. , 9410 Keywest Avenue, Rockville, MD 20850, USA 1 Department of Psychiatry, UMDNJ-Robert Wood Johnson Medical School , Piscataway, NJ 08854, USA 2 Department of Microbiology, University of Saskatchewan , Saskatoon, SK S7N 5E5, Canada *To whom correspondence should be addressed. Tel: +1 306 966 4308; Fax: +1 306 966 4311; Email - Eukaryotic genes encoding ubiquitin-congugating enzyme (Ubc)-like proteins have been isolated from both human and yeast cells. The CROC-1 gene was isolated by its ability to transactivate c-fos expression in cell culture through a tandem repeat enhancer sequence. The yeast MMS2 gene was cloned by its ability to complement the methyl methanesulfonate sensitivity of the mms2-1 mutant and was later shown to be involved in DNA post-replication repair. We report here the identification of a human MMS2 (hMMS2) cDNA encoding a novel human Ubc-like protein. hMMS2 and CROC-1 share >90% amino acid sequence identity, but their DNA probes hybridize to distinct transcripts. hMMS2 and CROC-1 also share ~ 50% identity and 75% similarity with the entire length of yeast Mms2. Unlike CROC-1, whose transcript appears to be elevated in all tumor cell lines examined, the hMMS2 transcript is only elevated in some tumor cell lines. Collectively, these results indicate that eukaryotic cells may contain a highly conserved family of Ubc-like proteins that play roles in diverse cellular processes, ranging from DNA repair to signal transduction and cell differentiation. The hMMS2 and CROC-1 genes are able to functionally complement the yeast mms2 defects with regard to sensitivity to DNA damaging agents and spontaneous mutagenesis. Conversely, both MMS2 and hMMS2 were able to transactivate a c-fosCAT reporter gene in Rat-1 cells in a transient co-transfection assay. We propose that either these proteins function in a common cellular process, such as DNA repair, or they exert their diverse biological roles through a similar biochemical interaction relative to ubiquitination. Ubiquitin (Ub) is a highly conserved 76 residue protein and is found in eukaryotic cells either as an unbound molecule or covalently joined to a variety of proteins (for reviews see13). Ub conjugation has been shown to participate in many eukaryotic metabolic processes, including ribosome biogenesis (4), mating type regulation (5), cell cycle control (6), DNA repair (7) and other responses (8). Ub is bound to the ubiquitin-activating enzyme (E1), which activates Ub and enables it to bind to the ubiquitin-conjugating enzyme (Ubc or E2). A single cysteine residue in a highly conserved region of Ubc is absolutely required to bind Ub via a thioester bond and attach it to the target molecule. In most cases, a third protein or protein complex is required as a ubiquitin ligase enzyme (E3) to select and polyubiquitinate target proteins for degradation (13,9). There are at least 13 different E2 enzymes in the yeast Saccharomyces cerevisiae, most of which confer distinct and non-overlapping functions, although some Ubc functions may be redundant, e.g. Ubc4 and Ubc5 (10) and Ubc6 and Ubc7 (11). All known eukaryotic E2 enzymes are highly conserved in their primary sequence, especially around the active Cys residue region (1,2) and their core tertiary structures are also conserved (1113). In contrast, E3 enzymes may consist of either a single polypeptide, such as Ubr1 (14), or a complex of several subunits, such as SCFCdc4 (15,16) or APCCdc20 (17,18), none of which share apparent sequence homology. However, all known E3 enzymes are able to form a complex with a specific E2 to target distinct protein substrates for degradation (1418). Recently, Ub-like proteins have been found in many eukaryotes. Like Ub, these proteins (e.g. Smt3 and Rub1 from yeast) also participate in post-translational modification of cellular proteins through biochemically conserved but genetically distinct E1 and E2 enzymes (for recent reviews see 19,20). Ubc9 conjugates Smt3 in vivo (21,22), which appears to be involved in subcellular localization of the target protein (23). Similarly, Ubc12 is required for Rub1 conjugation to Cdc53, which is probably involved in cell cycle regulation (24,25). Interestingly, eukaryotic genes encoding Ubc-like proteins were also identified recently. CROC-1 was isolated from a human cDNA library by its ability to transactivate a c-fos promoter via the 8 bp tandem repeat enhancer element (26). The same gene (also known as UEV-1 and CIR1) was subsequently isolated and characterized by differential display techniques and found to be decreased in HT-29-M6 human colon carcinoma cells undergoing differentiation (27) and to be increased in SV40-transformed human embryonic kidney cells undergoing immortalization (28). Furthermore, overexpression of the CROC-1 gene in HT-29-M6 cells inhibits the activity of the mitotic kinase cdk1 and cell differentiation (27). These results collectively suggest a role for CROC-1 in mammalian cell proliferation and differentiation. However, it remains to be determined if the biological effects of CROC-1 are solely due to its transactivation activity of c-fos (26) or inhibition of cdk1 activity (27). In parallel, an S.cerevisiae homolog of CROC-1, denoted MMS2, was independently isolated (29) by complementation of the mms2-1 mutant (30), which was sensitive to killing by methyl methanesulfonate (MMS), a DNA alkylating agent. The yeast MMS2 gene is involved in protection of cells from a variety of DNA damage, since disruption of the MMS2 gene not only results in an increased killing by MMS and UV irradiation, but also dramatically increases the spontaneous mutation rate (29). Genetic analyses indicate that MMS2 functions in the error-free post-replication repair (PRR) branch of the RAD6 pathway (29), but its exact biochemical activity remains to be determined. Although CROC-1 and Mms2 share significant amino acid sequence homology with Ubcs (up to P = 107) and their size and predicted secondary and tertiary structures resemble E2 enzymes (27), CROC-1 and Mms2 lack the active site cysteine residue within the highly conserved Ubc motif; indeed, neither CROC-1 nor Mms2 appears to confer Ubc activity in an in vitro assay (27,29). At the amino acid sequence level, CROC-1 and Mms2 are much more homologous with each other than to Ubcs (29), suggesting that they may form a separate Ubc-like protein family. We report here the isolation and characterization of a human cDNA, which we denote hMMS2, encoding a second MMS2 homolog and demonstrate that the function of hMMS2, CROC-1 and MMS2 are conserved in heterologous hosts, indicating that these genes may represent a novel gene family with a broad spectrum of evolutionarily conserved functions. MATERIALS AND METHODS Saccharomyces cerevisiae strains Haploid yeast strains DBY747 (MATa his3-D 1 leu2-3,112 ura3-52 trp1-289) and FY86 (...truncated)