Assay by inhibition of repair to measure O⁶‐methylguanine in DNA

Abstract

A procedure for measuring the level of O6-methylguanine (O6-meG) in DNA is described. Repair of 32P-oligodeoxynucleotides containing O6-meG adducts by O6-alkylguanine alkyltransferase (AGT) was performed in the presence of different quantities of DNA containing unknown concentrations of O6-meG. Each methylated DNA sample inhibited the repair of oligodeoxynucleotide substrate to an extent dependent upon O6-meG concentration. Each DNA sample tested at different concentrations in the assay therefore had a characteristic inhibition curve and could be compared to the curves generated using reference DNA samples of known O6-meG concentration. We report the method of calculation of the O6-meG level in a given DNA sample by comparison of its inhibition curve with that of reference DNAs. This method of calculation does not require a knowledge of the exact quantity of the labelled substrate or AGT used. The method requires only 0.1-10 micrograms of DNA, with a limit of detection of 0.8 fmol of O6-meG per microgram of DNA.

Proofreading Activity of Pfu Thermostable DNA Polymerase on a 6‐O‐Methylguanine‐Containing Template Monitored by ESI‐FTICR Mass Spectrometry

“…One commonly studied DNA adduct is 6‐ O ‐methyl (6‐ O ‐me) deoxyguanine (dG), formed in vivo by methylnitrosurea and tobacco smoke 3. 5 This is considered to be an important precarcinogenic lesion 2. 6‐ O ‐me dG is well known to induce a base mismatch, with thymine at the complementary position, by mammalian polymerase β, T4 and the Klenow DNA polymerase 6.…”