O6-Alkylguanine-DNA Alkyltransferases Repair O 6-Methylguanine in DNA with Michaelis-Menten-like Kinetics

Aviva S. Meyer, Melodie D. McCain, Qingming Fang, Anthony Pegg, Thomas Spratt

Research output: Contribution to journalArticle

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Abstract

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs O 6-methylguanine (O6mG) 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′-32P]-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 O6mG, annealed to its complementary strand. The reactions, conducted at 25 °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 kobs = kinact[AGT] o/(Ks + [AGT]0). The Ks values for hAGT of 81-91 nM are 10-fold lower than the dissociation constants of hAGT (C145S) to unmodified and O6mG-containing DNA obtained by EMSA and indicate that AGT has a preference for binding to O6mG in DNA. The proteins reacted with DNA in which Y = O6mG and Z = G faster than Y = G and Z = O6mG due to an approximately 10-fold increase in k inact. These results suggest that the sequence specificity in the repair of O6mG is manifested in the methyl transfer not in the O 6mG recognition step.

Original languageEnglish (US)
Pages (from-to)1405-1409
Number of pages5
JournalChemical research in toxicology
Volume16
Issue number11
DOIs
StatePublished - Nov 1 2003

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DNA Repair
Repair
Kinetics
DNA
Oligodeoxyribonucleotides
Ion Exchange
DNA alkyltransferase
O-(6)-methylguanine
Cysteine
Rate constants
Ion exchange
Proteins
High Pressure Liquid Chromatography

All Science Journal Classification (ASJC) codes

  • Toxicology

Cite this

@article{97b1d94e57a14eefb1b1c1f918f274d2,
title = "O6-Alkylguanine-DNA Alkyltransferases Repair O 6-Methylguanine in DNA with Michaelis-Menten-like Kinetics",
abstract = "O6-Alkylguanine-DNA alkyltransferase (AGT) repairs O 6-methylguanine (O6mG) 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′-32P]-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 O6mG, annealed to its complementary strand. The reactions, conducted at 25 °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 kobs = kinact[AGT] o/(Ks + [AGT]0). The Ks values for hAGT of 81-91 nM are 10-fold lower than the dissociation constants of hAGT (C145S) to unmodified and O6mG-containing DNA obtained by EMSA and indicate that AGT has a preference for binding to O6mG in DNA. The proteins reacted with DNA in which Y = O6mG and Z = G faster than Y = G and Z = O6mG due to an approximately 10-fold increase in k inact. These results suggest that the sequence specificity in the repair of O6mG is manifested in the methyl transfer not in the O 6mG recognition step.",
author = "Meyer, {Aviva S.} and McCain, {Melodie D.} and Qingming Fang and Anthony Pegg and Thomas Spratt",
year = "2003",
month = "11",
day = "1",
doi = "10.1021/tx0341254",
language = "English (US)",
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pages = "1405--1409",
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O6-Alkylguanine-DNA Alkyltransferases Repair O 6-Methylguanine in DNA with Michaelis-Menten-like Kinetics. / Meyer, Aviva S.; McCain, Melodie D.; Fang, Qingming; Pegg, Anthony; Spratt, Thomas.

In: Chemical research in toxicology, Vol. 16, No. 11, 01.11.2003, p. 1405-1409.

Research output: Contribution to journalArticle

TY - JOUR

T1 - O6-Alkylguanine-DNA Alkyltransferases Repair O 6-Methylguanine in DNA with Michaelis-Menten-like Kinetics

AU - Meyer, Aviva S.

AU - McCain, Melodie D.

AU - Fang, Qingming

AU - Pegg, Anthony

AU - Spratt, Thomas

PY - 2003/11/1

Y1 - 2003/11/1

N2 - O6-Alkylguanine-DNA alkyltransferase (AGT) repairs O 6-methylguanine (O6mG) 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′-32P]-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 O6mG, annealed to its complementary strand. The reactions, conducted at 25 °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 kobs = kinact[AGT] o/(Ks + [AGT]0). The Ks values for hAGT of 81-91 nM are 10-fold lower than the dissociation constants of hAGT (C145S) to unmodified and O6mG-containing DNA obtained by EMSA and indicate that AGT has a preference for binding to O6mG in DNA. The proteins reacted with DNA in which Y = O6mG and Z = G faster than Y = G and Z = O6mG due to an approximately 10-fold increase in k inact. These results suggest that the sequence specificity in the repair of O6mG is manifested in the methyl transfer not in the O 6mG recognition step.

AB - O6-Alkylguanine-DNA alkyltransferase (AGT) repairs O 6-methylguanine (O6mG) 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′-32P]-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 O6mG, annealed to its complementary strand. The reactions, conducted at 25 °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 kobs = kinact[AGT] o/(Ks + [AGT]0). The Ks values for hAGT of 81-91 nM are 10-fold lower than the dissociation constants of hAGT (C145S) to unmodified and O6mG-containing DNA obtained by EMSA and indicate that AGT has a preference for binding to O6mG in DNA. The proteins reacted with DNA in which Y = O6mG and Z = G faster than Y = G and Z = O6mG due to an approximately 10-fold increase in k inact. These results suggest that the sequence specificity in the repair of O6mG is manifested in the methyl transfer not in the O 6mG recognition step.

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SN - 0893-228X

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