Repair of O4-Alkylthymine by O6-Alkylguanine-DNA Alkyltransferases

O6-Alkylguanine-DNA alkyltransferase (AGT) plays a major role in repair of the cytotoxic and mutagenic lesion O6-methylguanine (m6G) in DNA. Unlike the Escherichia coli alkyltransferase Ogt that also repairs O4-methylthymine (m4T) efficiently, the human AGT (hAGT) acts poorly on m4T. Here we made several hAGT mutants in which residues near the cysteine acceptor site were replaced by corresponding residues from Ogt to investigate the basis for the inefficiency of hAGT in repair of m4T. Construct hAGT-03 (where hAGT sequence -V149CSSGAVGN157- was replaced with the corresponding Ogt -I143GRNGTMTG151-) exhibited enhanced m4T repair activity in vitro compared with hAGT. Three AGT proteins (hAGT, hAGT-03, and Ogt) exhibited similar protection from killing by N-methyl-N′-nitro-N-nitrosoguanidine and caused a reduction in m6G-induced G:C to A:T mutations in both nucleotide excision repair (NER)-proficient and -deficient Escherichia coli strains that lack endogenous AGTs. hAGT-03 resembled Ogt in totally reducing the m4T-induced T:A to C:G mutations in NER-proficient and -deficient strains. Surprisingly, wild type hAGT expression caused a significant but incomplete decrease in NER-deficient strains but a slight increase in T:A to C:G mutation frequency in NER-proficient strains. The T:A to C:G mutations due to O4-alkylthymine formed by ethylating and propylating agents were also efficiently reduced by either hAGT-03 or Ogt, whereas hAGT had little effect irrespective of NER status. These results show that specific alterations in the hAGT active site facilitate efficient recognition and repair of O4-alkylthymines and reveal damage-dependent interactions of base and nucleotide excision repair.