MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway

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.

Figure 1

Physical characterization of the S. cerevisiae MMS2 gene. (A) Mapping and disruption of the MMS2 gene. A subclone containing the 3-kb XbaI–HindIII fragment enables (+) the mms2-1 mutant to grow on YPD plates containing 0.4% MMS. Further deletions to the NcoI site from either end abolished (−) the MMS2 function. Either a URA3 or a LEU2 fragment was inserted at the NcoI site to construct the mms2∷URA3 and mms2∷LEU2 disruption cassettes. (B) Killing of DBY747 (wt), WX17–4a (mms2-1), and SBU (mms2) in a liquid culture containing 0.3% MMS. (C) The nucleotide and deduced amino acid sequences of the MMS2 gene (GenBank accession no. U66724). Exons are in boldface. Lowercase indicates intron sequences. Consensus sequences within the intron are underlined. The translation initiation site for MAD1 and the transcriptional termination site for SNR10 are indicated with an arrow for direction. A C-to-T transition found in the mms2-1 mutation at nucleotide 303 is marked. (D) Amino acid sequence alignments of Mms2 with two yeast Ubc proteins, Rad6 (Ubc2) and Ubc4. Residues shared by two or more proteins are highlighted. (E) Amino acid sequence alignment of Mms2 with Croc1. Residues in Croc1 identical to Mms2 are highlighted.

Figure 2

Epistatic analyses of mms2 with radiation repair pathway mutations with respect to UV sensitivity. (A–D) □, DBY747 (wt); ▪, SBU (mms2). (A) mms2 and rad6. ○, WXY9376 (rad6Δ); •, SBU6L (rad6Δ mms2). (B) mms2 and rad18Δ. ○, WXY9326 (rad18Δ); •, SBU18T (rad18Δ mms2). (C) mms2 and rad4Δ. ○, WXY9394 (rad4Δ); •, SBL4U (rad4Δ mms2). (D) mms2 and rad50Δ. ○, WXY9579 (rad50Δ); •, SBU50h (rad50Δ mms2). The results are an average of two to four independent experiments.

Figure 3

mms2 is synergistic to rev3 with respect to both UV (A) and MMS (B) sensitivity. □, DBY747 (wild type); ▪, SBU (mms2); ○, WXY9382 (rev3Δ); •, SBUr3L (rev3Δmms2). The results are an average of two independent experiments.

Figure 4

mms2 is hypostatic to rad6_Δ1–9 with respect to both UV (A) and MMS (B) sensitivity. □, DBY747 (wild type); ▪, SBU (mms2); ○, WXY9376/pSCW-rad6_Δ1–9 (rad6_Δ1–9); •, SBU6L/pSCW-rad6_Δ1–9 (mms2 rad6_Δ1–9). The results are an average of two independent experiments.

Figure 5

Schematic diagram of possible Rad6-mediated metabolic pathways. Rad6 forms distinct complexes with either Ubr1 or Rad18; the Rad6–Rad18 complex is proposed to be responsible for DNA repair (–21). The DNA repair component consists of an N-terminal-dependent error-free PRR pathway and an N-terminal-independent error-prone mutagenesis pathway. Mms2, PCNA, Polδ, and Rad30 are proposed to participate in the error-free PRR, whereas Rev1, -3, and -7 are responsible for mutagenesis.