A role for Saccharomyces cerevisiae Tpa1 protein in direct alkylation repair [Enzymology]

November 7th, 2014 by Shivange, G., Kodipelli, N., Monisha, M., Anindya, R.

Alkylating agents induce cytotoxic DNA base-adducts. In this report we provide evidence to suggest, for the first time, that S. 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 methylamethane sulfonate (MMS) sensitivity in Saccharomyces cerevisiae. Using purified Tpa1 we demonstrate that Tpa1 repairs both single and double-stranded methylated DNA. Tpa1 is a member of Fe(II) and 2-oxoglutarate-dependent dioxygenase family and we show that mutation of the amino acid residues involved in cofactor binding abolishes Tpa1 DNA repair activity. Deletion of TPA1 along with base excision repair (BER) pathway DNA glycosylase MAG1 renders the tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that trans-lesion synthesis DNA polymerases Polζ (REV3) plays key role in tolerating DNA methyl-base lesions and tpa1Δmag1revΔ3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate synergism between the BER pathway and direct alkylation repair by Tpa1 in Saccharomyces cerevisiae. We conclude that Tpa1 is hitherto unidentified DNA repair protein in yeast and plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity.