| Abstract
| - O6-Alkylguanine-DNA alkyltransferase (AGT) repairs DNA by transferring the methyl groupfrom the 6-position of guanine to a cysteine residue on the protein. We previously found that the Escherichiacoli 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-strandedpentadecadeoxynucleotides containing analogues of O6mG. The second-order rate constants were in thefollowing order (×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 bindingaffinity to hAGT. Oligodeoxynucleotides containing O6mG, S6mG and O6m3DG bound to AGT in thepresence of competitor DNA with Kd values from 5 to 20 μM, while those containing G, S6mH, O6m1DG, and O6m7DG did not (Kd> 200 μM). These results indicate that the 1-, N2-, and 7- positions ofO6mG 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 reasonada AGT reacts with O4mT faster than hAGT.
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