title: O6-alkylguanine-DNA alkyltransferases repair O6-methylguanine in DNA with Michaelis-Menten-like kinetics.
abstract: O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs O(6)-methylguanine (O(6)mG) by transferring the methyl group from the DNA to a cysteine residue on the protein. The kinetics of this reaction was examined by reacting an excess of AGT (0-300 nM) with [5'-(32)P]-labeled oligodeoxynucleotides (0.5 nM) of the sequence 5'-CGT GGC GCT YZA GGC GTG AGC-3' in which Y or Z was G or O(6)mG, annealed to its complementary strand. The reactions, conducted at 25 degrees C, were quenched by the addition of 0.1 N NaOH at various times, and the extents of reaction were monitored by ion exchange HPLC with radiochemical detection. The time courses followed first-order kinetics. The first-order rate constants were plotted against the initial concentration of AGT and fitted to the hyperbolic equation k(obs) = k(inact)[AGT](0)/(K(S) + [AGT](0)). The K(S) values for hAGT of 81-91 nM are 10-fold lower than the dissociation constants of hAGT (C145S) to unmodified and O(6)mG-containing DNA obtained by EMSA and indicate that AGT has a preference for binding to O(6)mG in DNA. The proteins reacted with DNA in which Y = O(6)mG and Z = G faster than Y = G and Z = O(6)mG due to an approximately 10-fold increase in k(inact). These results suggest that the sequence specificity in the repair of O(6)mG is manifested in the methyl transfer not in the O(6)mG recognition step.
authors: Institute for Cancer Prevention, American Health Foundation Cancer Center, One Dana Road, Valhalla, New York 10595, USA.
authors: Aviva S
authors: AS
authors: Meyer
authors: Melodie D
authors: MD
authors: McCain
authors: Qingming
authors: Q
authors: Fang
authors: Anthony E
authors: AE
authors: Pegg
authors: Thomas E
authors: TE
authors: Spratt
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D045643:DNA Repair Enzymes / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006147:Guanine / Q000031:analogs & derivatives* / Q000502:physiology*; D007700:Kinetics; D008745:Methylation / Q000187:drug effects; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000502:physiology*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects / Q000502:physiology; D013696:Temperature; D013997:Time Factors
chemical_list: D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D006147:Guanine; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D045643:DNA Repair Enzymes