MMS1 protects against replication-dependent DNA damage in Saccharomyces cerevisiae.

A series of yeast mutants were isolated that are sensitive to killing by the monofunctional DNA-alkylating agent methyl methanesulfonate (MMS) but not by UV or X-radiation. We have cloned and characterized one of the corresponding genes, MMS1, and show that the mms1 Delta mutant is dramatically sensitive to killing by MMS and mildly sensitive to UV radiation. mms1 Delta mutants display an elevated level of spontaneous DNA damage and genomic instability. Furthermore, the mms1 Delta cells are sensitive to killing by conditions that induce replication-dependent double-strand breaks, such as treatment with camptothecin, and incubation of a cdc2-2 strain at the restrictive temperature. rad52 Delta is epistatic to mms1 Delta for MMS and camptothecin sensitivity, indicating that Mms1 acts in concert with Rad52. However, unlike mutants of the RAD52 group, mms1 Delta cells are not sensitive to gamma-rays, which induce double-strand breaks independently of DNA replication. Together these results suggest a role for an Mms1-dependent, Rad52-mediated, pathway in protecting cells against replication-dependent DNA damage.

The Saccharomyces cerevisiae mre11(ts) allele confers a separation of DNA repair and telomere maintenance functions.

The yeast Mre11 protein participates in important cellular functions such as DNA repair and telomere maintenance. Analysis of structure-function relationships of Mre11 has led to identification of several separation-of-function mutations as well as N- and C-terminal domains essential for Mre11 meiotic and mitotic activities. Previous studies have established that there is a strong correlation between Mre11 DNA repair and telomere maintenance functions and that Mre11-Rad50-Xrs2 complex formation appears to be essential for both of these activities. Here we report that the mre11(ts) allele, previously shown to cause temperature-dependent defects in DNA repair and meiosis, confers a temperature-independent telomere shortening, indicating that mre11(ts) is a separation-of-function mutation with respect to DNA repair and telomere maintenance. In a yeast two-hybrid system, Mre11(ts) fails to form a homodimer or interact with Rad50 and Xrs2 irrespective of experimental temperatures. These observations collectively suggest that the Pro(162)Ser substitution in Mre11(ts) confers a novel separation of Mre11 mitotic functions. Moreover, we observed that while overexpression of the 5'-3' exonuclease gene EXO1 partially complements the MMS sensitivity of mre11, rad50, and xrs2 null mutants, it has no effect on telomere shortening in these strains. This result provides additional evidence on possible involvement of distinctive mechanisms in DNA repair and telomere maintenance by the Mre11-Rad50-Xrs2 complex.

Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae.

Evidence obtained from recent studies supports the existence of an error-free postreplication repair (PRR) and a mutagenesis pathway within the Saccharomyces cerevisiae RAD6 DNA repair group. The MMS2 gene is the only known yeast gene involved in error-free PRR that, when mutated, significantly increases the spontaneous mutation rate. In this study, the mutational spectrum of the mms2 mutator was determined and compared to the wild type strain. In addition, mutagenenic effects and genetic interactions of the mms2 mutator and rev3 anti-mutator were examined with respect to forward mutations, frameshift reversions as well as amber and ochre suppressions. It was concluded from these results that the mms2 mutator phenotype is largely dependent on the functional REV3 gene. The synergistic effects of mms2 and rev3 mutations towards killing by a variety of DNA-damaging agents ruled out the possibility that MMS2 simply acts to suppress REV3 activity and favored the hypothesis that MMS2 and REV3 form two alternative subpathways within the RAD6 DNA repair pathway. Taken together, we propose that two pathways represented by MMS2 and REV3 deal with a similar range of endogenous and environmental DNA damage but with different biological consequences, namely, error-free repair and mutagenesis, respectively.

REV3 is required for spontaneous but not methylation damage-induced mutagenesis of Saccharomyces cerevisiae cells lacking O6-methylguanine DNA methyltransferase.

O6-methylguanine (O6-MeG) DNA methyltransferase (MTase) removes the methyl group from a DNA lesion and directly restores DNA structure. It has been shown previously that bacterial and yeast cells lacking such MTase activity are not only sensitive to killing and mutagenesis by DNA methylating agents, but also exhibit an increased spontaneous mutation rate. In order to understand molecular mechanisms of endogenous DNA alkylation damage and its effects on mutagenesis, we determined the spontaneous mutational spectra of the SUP4-o gene in various Saccharomyces cerevisiae strains. To our surprise, the mgt1 mutant deficient in DNA repair MTase activity exhibited a significant increase in G:C-->C:G transversions instead of the expected G:C-->A:T transition. Its mutational distribution strongly resembles that of the rad52 mutant defective in DNA recombinational repair. The rad52 mutational spectrum has been shown to be dependent on a mutagenesis pathway mediated by REV3. We demonstrate here that the mgt1 mutational spectrum is also REV3-dependent and that the rev3 deletion offsets the increase of the spontaneous mutation rate seen in the mgt1 strains. These results indicate that the eukaryotic mutagenesis pathway is directly involved in cellular processing of endogenous DNA alkylation damage possibly by the translesion bypass of lesions at the cost of G:C-->C:G transversion mutations. However, the rev3 deletion does not affect methylation damage-induced killing and mutagenesis of the mgt1 mutant, suggesting that endogenous alkyl lesions may be different from O6-MeG.

Identification, chromosomal mapping and tissue-specific expression of hREV3 encoding a putative human DNA polymerase zeta.

The Saccharomyces cerevisiae REV3 gene encodes the catalytic subunit of a non-essential DNA polymerase zeta, which is required for mutagenesis. The rev3 mutants significantly reduce both spontaneous and DNA damage-induced mutation rates. We have identified human cDNA clones from two different libraries whose deduced amino acid sequences bear remarkable homology to the yeast Rev3, and named this gene hREV3. The hREV3 gene was mapped to chromosome 1p32-33 by fluorescence in situ hybridization. The hREV3 encodes an mRNA of >10 kb, and its expression varies in different tissues and appears to be elevated in some but not all of the tumor cell lines we have examined. In light of recent reports of a putative mouse REV3, these results indicate that mammalian cells may also contain a mutagenic pathway which aids in cell survival at the cost of increased mutation.

Mutagenicity and toxicity of the DNA alkylation carcinogens 1,2-dimethylhydrazine and azoxymethane in Escherichia coli and Salmonella typhimurium.

DNA alkylating agents such as 1,2-dimethylhydrazine (SDMH) and azoxymethane (AOM) are potent carcinogens and are widely used to induce colon tumors in experimental animals. However, standard bacterial mutagenesis assays have failed to detect the mutagenic effects of these chemicals. Using derivatives of a set of Escherichia coli test strains developed by Cupples and Miller (Proc. Natl. Acad. Sci. USA, 86, 5345, 1989), we hve demonstrated that under two conditions, SDMH and AOM induced point mutations by several-fold in a dose-dependent manner: (i) of six possible base substitutions, they only induced GC-->AT transitions; and (ii) the cells must be deficient in O6-methylguanine (O6MeG) DNA methyltransferase (MTase) activity. SDMH and AOM up to 200 microg/ml were unable to induce His+ revertants in a Salmonella Ames test strain TA1535 (GC-->AT); however, in the absence of mammalian S9 extract, His+ revertants increased up to 55-fold upon treatment of an isogenic Salmonella strain deficient in MTase activity. These results indicate that SDMH and AOM are indeed bacterial mutagens and that lesions induced by them are the target of DNA repair MTases, which probably include mutagenic and carcinogenic lesions such as O6MeG. Furthermore, variable responses of bacterial species to SDMH- and AOM-induced mutagenicity suggests a difference either in the metabolism of potential mutagens or in the repair of specific lesions. Since O6MeG is not only a mutagenic lesion but also a lethal lesion if left unrepaired, we compared the mutagenicity and toxicity of SDMH and AOM with an SN-type methylating carcinogen. N-methyl-N'-nitro-N-nitrosoguanidine, and conclude that SDMH and AOM are weak bacterial mutagens.

DNA mismatch repair mutants do not increase N-methyl-N'-nitro-N-nitrosoguanidine tolerance in O6-methylguanine DNA methyltransferase-deficient yeast cells.

Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) produces, among other lesions, mutagenic and carcinogenic lesions such as O6-methylguanine (O6MeG) and O4-methylthymine in DNA. An O6MeG DNA methyl-transferase (MTase) specifically and efficiently repairs such lesions. MTase-deficient bacterial, yeast and mammalian cells exhibit increased sensitivity not only to MNNG-induced mutagenesis, but also to MNNG-induced killing, suggesting that O6MeG-type lesions are also lethal to the cells. The lethal effect caused by O6MeG is not clear. Results from several recent experiments indicate that some MNNG-tolerant cell lines exhibit a loss of DNA mismatch binding/repair activity, suggesting that functional mismatch repair is probably responsible for the cellular sensitivity to DNA methylating agents. We tested this abortive O6MeG-T mismatch repair hypothesis in a well-defined lower eukaryote, Saccharomyces cerevisiae. We found that while mgt1-deleted MTase-deficient yeast strains are hypersensitive to MNNG-induced killing, combination of this mutation with any of the mlh1, msh2 or pms1 mutations did not render cells more tolerant to killing. msh3 mutation also did not rescue MNNG-induced genotoxicity. Furthermore, through the isolation and characterization of MNNG-tolerant cell lines from the MTase-deficient mutants we demonstrated that a DNA mismatch repair defect is neither sufficient nor required for this process. Since both DNA repair MTases and mismatch repair proteins are highly conserved between yeast and mammalian cells, our results could suggest alternative mechanisms in the cellular tolerance to O6MeG-induced killing.

Expression of the human MGMT O6-methylguanine DNA methyltransferase gene in a yeast alkylation-sensitive mutant: its effects on both exogenous and endogenous DNA alkylation damage.

Common Mer- cell lines deficient in O6-methylguanine DNA methyltransferase (MTase) activity probably result from the down-regulation of, rather than mutations in, the MGMT gene. However, the down-regulation of other unrelated genes was also observed in some of these cell lines, making it difficult to determine the precise functions of the MGMT MTase gene. To study the biological function of human MGMT MTase, we seek to utilize a newly created yeast mgt1 mutant deficient in the DNA repair MTase activity. The human MGMT cDNA was cloned into yeast expression vectors so that the MGMT gene is under the control of either an inducible GAL1 promoter or a constitutive ADH1 promoter. Upon galactose induction, the PGAL1-MGMT transformant had about 40-fold MTase activity compared to the wild-type strain. MGMT overexpression protected the yeast mgt1 mutant against alkylation-induced killing and mutation. Limited expression of the MGMT gene in the mgt1 mutant still provides significant alkylation resistance, albeit at a reduced level. The yeast mgt1 mutants increase spontaneous mutation rate, whereas constitutive expression of the MGMT gene lowered the spontaneous mutation rate in the mgt1 mutant to the wild-type level. We suggest that MGMT MTase may play the same role in human cells as the MGT1 MTase in yeast cells. Thus our results demonstrate that the human MGMT gene functionally complements the yeast MTase-deficient mutant in the protection against exogenous and endogenous DNA alkylation damage, which provides a useful tool for the study of in vivo mammalian MTase functions.

UBP5 encodes a putative yeast ubiquitin-specific protease that is related to the human Tre-2 oncogene product.

A gene from chromosome V of the yeast Saccharomyces cerevisiae has been cloned and sequenced. The deduced amino acid sequence encoded by this gene is similar to several ubiquitin-specific proteases from yeast, especially at the highly conserved domain. It is thus named UBP5. UBP5 is also closely related to the human Tre-2 and the mouse Unp oncogene products. This study adds a new member to the ubiquitin protease family and suggests that alteration of ubiquitin protease activity may result in cancer in mammals. However, disruption of the UBP5 gene in a haploid strain did not result in a noticeable phenotypic alteration.