Preparation of Covalently Linked Complexes Between DNA and <i>O</i>⁶‑Alkylguanine-DNA Alkyltransferase Using Interstrand Cross-Linked DNA

<i>O</i>⁶-alkylguanine-DNA alkyltransferases (AGT) are responsible for the removal of alkylation at both the <i>O</i>⁶ atom of guanine and <i>O</i>⁴ atom of thymine. AGT homologues show vast substrate differences with respect to the size of the adduct and which alkylated atoms they can restore. The human AGT (hAGT) has poor capabilities for removal of methylation at the <i>O</i>⁴ atom of thymidine, which is not the case in most homologues. No structural data are available to explain this poor hAGT repair. We prepared and characterized <i>O</i>⁶G-butylene-<i>O</i>⁴T (XLGT4) and <i>O</i>⁶G-heptylene-<i>O</i>⁴T (XLGT7) interstrand cross-linked (ICL) DNA as probes for hAGT and the Escherichia coli homologues, OGT and Ada-C, for the formation of DNA-AGT covalent complexes. XLGT7 reacted only with hAGT and did so with a cross-linking efficiency of 25%, while XLGT4 was inert to all AGT tested. The hAGT mediated repair of XLGT7 occurred slowly, on the order of hours as opposed to the repair of <i>O</i>⁶-methyl-2′-deoxyguanosine which requires seconds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the repair reaction revealed the formation of a covalent complex with an observed migration in accordance with a DNA-AGT complex. The identity of this covalent complex, as determined by mass spectrometry, was composed of a heptamethylene bridge between the <i>O</i>⁴ atom of thymidine (in an 11-mer DNA strand) to residue Cys145 of hAGT. This procedure can be applied to produce well-defined covalent complexes between AGT with DNA

Altering Residue 134 Confers an Increased Substrate Range of Alkylated Nucleosides to the E. coli OGT Protein

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