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MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication
S Broomfield1, B L Chow, W Xiao
1Department of Microbiology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK Canada S7N 5E5.
Abstract:
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 rad6Delta1-9 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, rad6Delta1-9 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.
Insights
The MMS2 gene in Saccharomyces cerevisiae is crucial for error-free DNA repair, functioning in a pathway parallel to mutagenesis. Its mutation reveals a new DNA repair mechanism, distinct from mutagenesis.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair Mechanisms
Background:
- The RAD6 epistasis group in Saccharomyces cerevisiae is complex, involving both error-free postreplication repair and mutagenesis pathways.
- Existing mutants like rad6 and rad18 affect both pathways, while rev3 mutants only impact mutagenesis, leaving the error-free pathway incompletely understood.
- A specific gene solely involved in error-free postreplication repair was previously unreported.
Purpose of the Study:
- To identify and characterize a gene involved exclusively in the error-free postreplication repair pathway within the RAD6 group.
- To elucidate the function of the MMS2 gene in DNA repair and mutagenesis in Saccharomyces cerevisiae.
Main Methods:
- Cloning of the MMS2 gene via functional complementation of the mms2-1 mutant.
- Sequence analysis of the MMS2 gene and its encoded protein.
- Genetic analysis of mms2 mutants, including epistasis studies with rad6, rad18, rev3, and pol30-46 mutants, and assessment of UV-induced mutagenesis and UV sensitivity.
Main Results:
- MMS2 encodes a protein homologous to ubiquitin-conjugating (Ubc) proteins but lacking Ubc activity.
- mms2 mutants are proficient in UV-induced mutagenesis but exhibit phenotypes similar to pol30-46, indicating a role in postreplication repair.
- Genetic interactions suggest MMS2 functions in the error-free postreplication repair pathway, parallel to the REV3 mutagenesis pathway.
Conclusions:
- The MMS2 gene is identified as a key component of the error-free DNA postreplication repair pathway in Saccharomyces cerevisiae.
- The mms2 null mutation, along with specific alleles of rad6 (rad6Δ1-9) and pol30 (pol30-46), defines this error-free repair mode.
- These mutations may also influence spontaneous and DNA damage-induced mutagenesis, suggesting crosstalk between repair pathways.