O⁶-Alkylguanine-DNA alkyltransferases (AGTs) are proteins responsible for the removal of mutagenic alkyl adducts at the O⁶-atom of guanine and O⁴-atom of thymine. In the current study we set out to understand the role of the Ser134 residue in the Escherichia coli AGT variant OGT on substrate discrimination. The S134P mutation in OGT increased the ability of the protein to repair both O⁶-adducts of guanine and O⁴-adducts of thymine. However, the S134P variant was unable, like wild-type OGT, to repair an interstrand cross-link (ICL) bridging two O⁶-atoms of guanine in a DNA duplex. When compared to the human AGT protein (hAGT), the S134P OGT variant displayed reduced activity towards O⁶-alkylation but a much broader substrate range for O⁴-alkylation damage reversal. The role of residue 134 in OGT is similar to its function in the human homolog, where Pro140 is crucial in conferring on hAGT the capability to repair large adducts at the O⁶-position of guanine. Finally, a method to generate a covalent conjugate between hAGT and a model nucleoside using a single-stranded oligonucleotide substrate is demonstrated.
Keywords: Bioorganic molecules; DNA interstrand cross-link; DNA repair; homology modeling; modified oligonucleotides; mutagenesis; substrate specificity.