Reaction and binding of oligodeoxynucleotides containing analogues of O⁶-methylguanine with wild-type and mutant human O⁶-alkylguanine-DNA alkyltransferase

O⁶-Alkylguanine-DNA alkyltransferase (AGT) repairs DNA by transferring the methyl group from the 6-position of guanine to a cysteine residue on the protein. We previously found that the Escherichia coli Ada protein makes critical interactions with O⁶-methylguanine (O⁶mG) at the N1- and O⁶- positions. Human AGT has a different specificity than the bacterial protein. We reacted hAGT with double-stranded pentadecadeoxynucleotides containing analogues of O⁶mG. The second-order rate constants were in the following order (x 10^-5 M^-1 s^-1): O⁶mG (1.4), O⁶-methylhypoxanthine (1.6) > Se⁶-methyl-6-selenoguanine (0.1) > S⁶-methyl-6-thioguanine (S⁶mG) (0.02) >> S⁶-methyl-6-thiohypoxanthine (S⁶mH), O⁶-methyl-1-deazaguanine (O⁶m1DG), O⁶-methyl-3-deazaguanine (O⁶m3DG), and O⁶-methyl-7- deazaguanine (O⁶m7DG) (all <0.0001). Electrophoretic mobility shift assays were carried out to determine the binding affinity to hAGT. Oligodeoxynucleotides containing O⁶mG, S⁶mG and O⁶m3DG bound to AGT in the presence of competitor DNA with K(d) values from 5 to 20 μM, while those containing G, S⁶mH, O⁶m1DG, and O⁶m7DG did not (K(d) > 200 μM). These results indicate that the 1-, N²-, and 7- positions of O⁶mG are critical in binding to hAGT, while the 3- and O⁶-positions are involved in methyl transfer. These results suggest that the active site of ada AGT is more flexible than hAGT and may be the reason ada AGT reacts with O⁴mT faster than hAGT.