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-V ¹⁴⁹CSSGAVGN¹⁵⁷- 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.