Molecular Cloning of a Gene,<i>DHS1</i>, Which Complements a Drug-hypersensitive Mutation of the Yeast<i>Saccharomyces cerevisiae</i>

Lee, Yun Sik; Shimizu, Jiro; Yoda, Koji; Yamasaki, Makari

doi:10.1271/bbb.58.391

Cited by 8 publications

(10 citation statements)

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“…Sequence analysis showed that C39 contained a region of the S. cerevisiae DHS1 gene (25). We cloned the DHS1 gene, and sequence analysis (GenBank accession no.…”

Section: Resultsmentioning

confidence: 99%

Identification and characterization of Saccharomyces cerevisiae EXO1 , a gene encoding an exonuclease that interacts with MSH2

Tishkoff

Boerger

Bertrand

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

355

353

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A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5-3 exonuclease. S. cerevisiae EXO1 interacted with both S. cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments. exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway. exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants.Genetic and biochemical studies have indicated eukaryotes contain a mismatch repair (MMR) pathway related to the bacterial MutHLS pathway (reviewed in refs. 1 and 2). However, recent evidence suggests that eukaryotic MMR is more complex. In Saccharomyces cerevisiae there are two MMR pathways that require MSH2, a MutS homologue that recognizes mispaired bases (3, 4). One is a single base substitution mispair pathway that requires a complex of MSH2 and the MutS homologue MSH6 (also called GTBP or p160 in humans) (1-3). There is also an insertion͞deletion mispair pathway that requires either a complex of MSH2 and MSH6 or a complex of MSH2 and MSH3, a third MutS homologue (1-3). Additionally, four S. cerevisiae MutL homologues have been identified, PMS1 (PMS2 in humans) and MLH1-MLH3; PMS1 and MLH1 function in MMR and have been shown to form a heterodimer (1, 2).In vitro studies in Escherichia coli have shown that the excision step of MMR can occur either 5Ј to 3Ј or 3Ј to 5Ј of the initiating nick and requires the combination of a helicase (UvrD) and one of three single-stranded DNA exonucleases (Exo I, Exo VII or RecJ) (reviewed in ref. 2). In eukaryotic MMR, proteins involved in excising the mispair have not been identified, although some candidates have been suggested. These include S. cerevisiae RAD27 (RTH1, YKL510), a 5Ј-3Ј exonuclease and flap endonuclease (5), and Schizosaccharomyces pombe EXO1 and its Drosophila homologue Tosca, which are members of the same family of endo-and exonucleases as RAD27 (6, 7).The importance of determining the mechanism of MMR is underscored by its association with hereditary nonpolyposis colorectal carcinoma (HNPCC) (reviewed in refs. 2 and 8). HNPCC is associated primarily with germ-line mutations in two human MMR genes, MSH2 and MLH1, whereas mutations in other MMR genes are rare (ref. 9; reviewed in refs. 2 and 8). Somatic mutations in MMR genes have been found in some sporadic tumors, suggesting some sporadic cancers could be due to acquired mutations in MMR genes (reviewed in ref. 8, and see ref. 10). However, not all of HNPCC or sporadic cancers with mutator phenotypes can be accounted for by known MMR genes (9, 10). Consequently, there has been interest in identifying additional MMR genes. Here we describe the use of a two-hybrid screen to identify proteins that interact with MSH2 and function...

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“…Sequence analysis showed that C39 contained a region of the S. cerevisiae DHS1 gene (25). We cloned the DHS1 gene, and sequence analysis (GenBank accession no.…”

Section: Resultsmentioning

confidence: 99%

Identification and characterization of Saccharomyces cerevisiae EXO1 , a gene encoding an exonuclease that interacts with MSH2

Tishkoff

Boerger

Bertrand

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

355

353

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“…EXO1 was originally cloned from S. cerevisiae as a gene that complemented a drug hypersensitive mutant, DHS1 (Lee et al 1994). It was also identified as a 5′-3′ exonuclease required for an in vitro recombination assay (Huang and Symington 1993).…”

Section: Introductionmentioning

confidence: 99%

EXO1 and MSH4 differentially affect crossing-over and segregation

Khazanehdari

Borts

2000

Chromosoma

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The 5'-3' exonuclease Exo1p from Saccharomyces cerevisiae is required for wild-type levels of meiotic crossing-over and normal meiotic chromosome segregation as is the meiosis-specific MutS homologue, Msh4p. Mutations in both genes reduce crossing-over by approximately two-fold, but deltamsh4 strains have significantly lower viability and a higher frequency of meiosis I non-disjunction. Epistasis analysis indicates a complex interaction between the two genes. Although crossing-over was not detectably lower in the double mutant, viability was significantly worse than either single mutant. Such a result suggests that the two genes are affecting meiotic viability by distinct mechanisms. We propose that deltaexo1 affects chromosome segregation by reducing crossing-over, while deltamsh4 affects both the frequency and distribution of crossovers. Mutation in EXO1 reduces gene conversion frequencies significantly at some but not all loci, suggesting that other enzymes are also involved in DNA resection. We propose that Exo1p plays an early role in establishing some recombination intermediates by generating single-stranded tails. The role of Msh4p is suggested to be in determining whether some recombination intermediates are resolved as crossover events and in generating crossover interference. The synergistic effect of deltaexo1deltamsh4 on spore viability suggests that the two genes have partially compensatory roles in a process affecting meiotic success.

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“…These include DHS1 (ORF YOR033C, chromosome XI), DIN7 (ORF YDR263C, chromosome IV), and YEN1 (ORF YER041W, chromosome V). The DHS1 gene was originally identified by genetic complementation of a drughypersensitive mutant (31). However, the protein sequence of Dhs1 (GenBank accession number S69545) is 219 amino acids shorter than the ORF identified by the S. cerevisiae genome sequencing project, and most of the conserved nuclease motifs are found within these 219 amino acids.…”

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confidence: 99%

Exonuclease I of Saccharomyces cerevisiae Functions in Mitotic Recombination In Vivo and In Vitro

Fiorentini

Huang

Tishkoff

et al. 1997

Molecular and Cellular Biology

183

134

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We previously described a 5-3 exonuclease required for recombination in vitro between linear DNA molecules with overlapping homologous ends. This exonuclease, referred to as exonuclease I (Exo I), has been purified more than 300-fold from vegetatively grown cells and copurifies with a 42-kDa polypeptide. The activity is nonprocessive and acts preferentially on double-stranded DNA. The biochemical properties are quite similar to those of Schizosaccharomyces pombe Exo I. Extracts prepared from cells containing a mutation of the Saccharomyces cerevisiae EXO1 gene, a homolog of S. pombe exo1, had decreased in vitro recombination activity and when fractionated were found to lack the peak of activity corresponding to the 5-3 exonuclease. The role of EXO1 on recombination in vivo was determined by measuring the rate of recombination in an exo1 strain containing a direct duplication of mutant ade2 genes and was reduced sixfold. These results indicate that EXO1 is required for recombination in vivo and in vitro in addition to its previously identified role in mismatch repair.The currently favored models for homologous recombination predict a requirement for nucleases at several steps in the recombination reaction (18,37,55). Endonucleases are envisioned to function as initiators of recombination and in the resolution of crossed-strand intermediates. Exonucleases are thought to process break sites to generate single-stranded DNA, the substrate for binding by homologous pairing proteins. Late steps in the reaction, such as the repair of heteroduplex DNA (mismatch correction), also require the activity of exonucleases. The importance of exonucleases in recombination has been clearly demonstrated in Escherichia coli, in which 5Ј-to-3Ј exonucleases are required for all homologous recombination pathways (30). In addition, the single-stranded exonucleases RecJ (5Ј), exonuclease VII (Exo VII) (5Ј and 3Ј), and Exo I (3Ј) are required for mismatch repair in vitro (8).In vivo studies of double-strand break (DSB) repair in Saccharomyces cerevisiae provide further support for the role of exonucleases in recombination. After HO endonuclease cleavage at the MAT locus during mating-type switching, the DNA is resected to produce a 3Ј tail on the distal side of the HO cut site (64). This single-strand tail is believed to invade the donor locus, thus initiating the transfer of information. Formation of the 3Ј single-stranded tail is thought to result from the activity of a double-stranded 5Ј-3Ј exonuclease, but it could arise from the combined activities of a helicase and single-stranded endonuclease or exonuclease (35). Although mutation of several known recombination genes prevents mating-type switching, none appears to block the exonucleolytic processing step (49, 64). However, the degradation of the 5Ј strand is slower in rad50 and xrs2 mutants, suggesting that these genes may encode or regulate the activity of a nuclease (21). Most meiotic recombination hot spots are cleaved by endonucleases during meiosis, and the resulting DSBs are ...

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Molecular Cloning of a Gene,DHS1, Which Complements a Drug-hypersensitive Mutation of the YeastSaccharomyces cerevisiae

Cited by 8 publications

References 3 publications

Identification and characterization of Saccharomyces cerevisiae EXO1 , a gene encoding an exonuclease that interacts with MSH2

Identification and characterization of Saccharomyces cerevisiae EXO1 , a gene encoding an exonuclease that interacts with MSH2

EXO1 and MSH4 differentially affect crossing-over and segregation

Exonuclease I of Saccharomyces cerevisiae Functions in Mitotic Recombination In Vivo and In Vitro

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