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

O-6-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 O-6-methylguanine (O(6)mG) at the N1- and O-6-positions. Human AGT has a different specificity than the bacterial protein. We reacted hAGT with double-stranded pentadecadeoxynucleotides containing analogues of O(6)mG. The second-order rate constants were in the following order (x 10(-5) M-1 s(-1)): O(6)mG (1.4), O-6-methylhypoxanthine (1.6) > Se-6-methyl-6-selenoguanine (0.1) > S-6-methyl-6-thioguanine (S(6)mG) (0.02) >> S-6-methyl-6-thiohypoxanthine (S(6)mH), O-6-methyl-1-deazaguanine (O(6)m1DG), O-6-methyl-3-deazaguanine (O(6)m3DG), and O-6-methyl-7-deazaguanine (O(6)m7DG) (all <0.0001). Electrophoretic mobility shift assays were carried out to determine the binding affinity to hAGT. Oligodeoxynucleotides containing O(6)mG, S(6)mG and O(6)m3DG bound to AGT in the presence of competitor DNA with K-d values from 5 to 20 mu M, while those containing G, S(6)mH, O(6)m1DG, and O(6)m7DG did not (K-d > 200 mu M). These results indicate that the 1-, N-2-, and 7- positions of O(6)mG are critical in binding to hAGT, while the 3- and O-6-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 O(4)mT faster than hAGT.