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A role for Saccharomyces cerevisiae Tpa1 protein in direct alkylation repair.

ABSTRACT: Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylated DNA. Tpa1 is a member of the Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that mutation of the amino acid residues involved in cofactor binding abolishes the Tpa1 DNA repair activity. Deletion of TPA1 along with the base excision repair pathway DNA glycosylase MAG1 renders the tpa1?mag1? double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Pol? (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1?mag1rev?3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate a synergism between the base excision repair pathway and direct alkylation repair by Tpa1 in S. cerevisiae. We conclude that Tpa1 is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity.

SUBMITTER: Shivange G

PROVIDER: S-EPMC4276862 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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A role for Saccharomyces cerevisiae Tpa1 protein in direct alkylation repair.

Shivange Gururaj G Kodipelli Naveena N Monisha Mohan M Anindya Roy R

The Journal of biological chemistry 20141107 52

Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylat ...[more]

PMID: 25381260

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Project description:spt23 plays multiple roles in the thermal tolerance of budding yeast. spt23 regulates unsaturated lipid acid (ULA) content in the cell, which can then significantly affect cellular thermal tolerance. Being a Ty suppressor, spt23 can also interact with transposons (Tys) that are contributors to yeast's adaptive evolution. Nevertheless, few studies have investigated whether and how much spt23 can exert its regulatory functions through transposons. In this study, expression quantitative trait loci (eQTL) analysis was conducted with thermal-tolerant Saccharomyces cerevisiae strains, and spt23 was identified as one of the most important genes in mutants. spt23-overexpression (OE), deletion (Del), and integrative-expressed (IE) strains were constructed. Their heat tolerance, ethanol production, the expression level of key genes, and lipid acid contents in the cell membranes were measured. Furthermore, LTR (long terminal repeat)-amplicon sequencing was used to profile yeast transposon activities in the treatments. The results showed the Del type had a higher survival rate, biomass, and ethanol production, revealing negative correlations between spt23 expression levels and thermal tolerance. Total unsaturated lipid acid (TULA) contents in cell membranes were lower in the Del type, indicating its negative association with spt23 expression levels. The Del type resulted in the lower richness and higher evenness in LTR distributions, as well as higher transposon activities. The intersection of 3 gene sets and regression analysis revealed the relative weight of spt23's direct and TY-induced influence is about 4:3. These results suggested a heat tolerance model in which spt23 increases cell thermal tolerance through transcriptional regulation in addition to spt23-transposon triggered unknown responses. KEY POINTS: • spt23 is a key gene for heat tolerance, important for LA contents but not vital. • Deletion of spt23 decreases in yeast's LTR richness but not in evenness. • The relative weight of spt23's direct and TY-induced influence is about 4:3.

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A role for Saccharomyces cerevisiae Tpa1 protein in direct alkylation repair.

Publications

A role for Saccharomyces cerevisiae Tpa1 protein in direct alkylation repair.

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