Molecular Characterization of Saccharomyces cerevisiae TPA1, an Oxidative Demethylase Involved in Direct Repair of DNA Alkylation Damage

Kodipelli, Naveena (2016) Molecular Characterization of Saccharomyces cerevisiae TPA1, an Oxidative Demethylase Involved in Direct Repair of DNA Alkylation Damage. PhD thesis, Indian Institute of Technology Hyderabad.

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Abstract

Alkylating agents induce cytotoxic DNA base-adducts. In this thesis I provide evidence to suggest 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 (MMS) sensitivity in Saccharomyces cerevisiae. Using purified Tpa1, I demonstrate that wild type Tpa1 repairs both single and double-stranded methylated DNA and mutation of the amino acid residues involved in cofactor binding abolishes Tpa1 DNA repair activity. In this thesis I also investigate genetic interaction of Tpa1. I also demonstrate that Tpa1 could complement alkB function and rescue the MMS sensitivity of alkB deficient E.coli strain, HK82. Experimental data proves that deletion of TPA1 along with base excision repair (BER) pathway DNA glycosylase MAG1 renders the tpa1 ∆ mag1 ∆ double mutant highly susceptible to methylation-induced toxicity. I further demonstrate that trans-lesion synthesis DNA polymerases Polζ (REV3) plays key role in tolerating DNA methyl-base lesions and tpa1∆mag1rev∆3 triple mutant is extremely susceptible to methylation-induced toxicity. These results indicate synergism between the BER pathway and direct alkylation repair by Tpa1 in Saccharomyces cerevisiae. I conclude that Tpa1 is hitherto unidentified DNA repair protein in yeast and plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity.

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