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 tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Polζ (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1Δmag1revΔ3 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.
Highlights
Fe(II)/2-oxoglutarate (2OG)-dependent dioxygenases repair alkyl-base lesions of DNA
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
When we examined the ability of the N-terminal domain (NTD) and C-terminal domain (CTD) in suppressing the MMS-sensitive phenotype of E. coli alkB strain HK82, we observed that the NTD successfully complemented alkylation repair protein B (AlkB) function but that the CTD could not, suggesting that the enzymatic activity of Tpa1 may be essential for complementing AlkB
Summary
Fe(II)/2-oxoglutarate (2OG)-dependent dioxygenases repair alkyl-base lesions of DNA. Results: Tpa is a Fe(II)/2OG-dependent dioxygenase and mediates alkyl-base repair in Saccharomyces cerevisiae, and deleting TPA1 with DNA glycosylase MAG1 had a synergistic effect on the susceptibility to methylation-induced toxicity. Significance: Our results provide the first evidence of direct alkylation repair by any Fe(II)/2OG-dependent dioxygenases in Saccharomyces cerevisiae. We provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa protein is involved in DNA alkylation repair. 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 conclude that Tpa is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity
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