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 (x10(-)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 Kd values from 5 to 20 microM, while those containing G, S6mH, O6m1DG, and O6m7DG did not (Kd > 200 microM). 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.
Spratt
T E
TE
American Health Foundation, Division of Pathology and Toxicology, Valhalla, New York 10595, USA.
Wu
J D
JD
Levy
D E
DE
Kanugula
S
S
Pegg
A E
AE
D001665:Binding Sites / Q000235:genetics; D004260:DNA Repair; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D006147:Guanine; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase