Abstract
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.
Highlights
The genetic information stored in DNA is under constant modification by endogenous and exogenous agents, including alkylating agents
Crystal structures of the human AGT in complex with DNA demonstrate that when bound by the protein, the alkyl appendage at both the O6 -atom of purines or O4 -atom of pyrimidines of modified DNA lies in a pocket formed by loop 135-144 [10,11]
We have previously shown that human AGT protein (hAGT) was capable of repairing intrastrand cross-linked (IaCL)
Summary
The genetic information stored in DNA is under constant modification by endogenous and exogenous agents, including alkylating agents. Crystal structures of the human AGT (hAGT) in complex with DNA demonstrate that when bound by the protein, the alkyl appendage at both the O6 -atom of purines or O4 -atom of pyrimidines of modified DNA lies in a pocket formed by loop 135-144 [10,11]. This loop contains 3 proline residues at positions 138, 140, 144.
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