title: A novel, sensitive assay for O6-methyl- and O6-ethylguanine in DNA, based on repair by the enzyme O6-alkylguanine-DNA-alkyltransferase in competition with an oligonucleotide containing O6-methylguanine.
abstract: A novel assay for O6-alkylguanine-type adducts in DNA is reported. It is based on the use of the suicide repair enzyme O6-alkylguanine-DNA-alkyltransferase (AGT) to repair such adducts in DNA in competition with an oligonucleotide containing a single residue of O6-methylguanine, end labeled to high specific activity. The stoichiometric mode of action of AGT results in decreased amounts of oligonucleotide being repaired in the presence of increasing levels of adducts in the competing DNA. The extent of oligonucleotide repair is determined by immunoprecipitation of the unrepaired form with rabbit antiserum directed against O6-alkyldeoxyguanosine and radiocounting. The amount of O6-alkylguanine in competing DNA is calculated by reference to a standard curve constructed using DNA of known alkylation. In view of its relatively wide spectrum of alkyl group specificity, use of AGT from rat liver permits the determination of both O6-methyl- and O6-ethylguanine (detection limits, 0.8 fmol and 3 fmol, respectively). On the other hand, the restricted specificity of Escherichia coli AGT to repair of O6-methylguanine makes the assay based on it specific for this type of lesion (detection limit, 0.5 fmol). The maximum amount of DNA which can be included in the assay is 15 micrograms and 10 micrograms for the rat liver and E. coli AGT-based assays, respectively, leading to a limit of sensitivity of 8 x 10(-8) mol O6-methylguanine/mol guanine (50 fmol/mg DNA) (both enzymes) and 3 x 10(-7) mol O6-ethylguanine/mol guanine (200 fmol/mg DNA) (rat liver AGT-based assay) and making this one of the most sensitive assays for these important precarcinogenic adducts. The new assay has been validated by assaying DNA from rat liver methylated in vivo with dimethylnitrosamine to a known extent and has been found to give results in close agreement with those of radioimmunoassay. Six h after i.p. administration of dimethylnitrosamine (0.01-1 mg/kg) to rats, O6-methylguanine was detectable by the competitive-repair assay in liver or lymphocyte DNA at levels of 0.14-14.4 mumol/mol guanine.
authors.affiliation: National Hellenic Research Foundation, Institute of Biological Research, Athens, Greece.
authors.forename: V L
authors.initials: VL
authors.lastname: Souliotis
authors.forename: S A
authors.initials: SA
authors.lastname: Kyrtopoulos
mesh_terms: D000818:Animals; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008780:Methyltransferases / Q000378:metabolism*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009841:Oligonucleotides / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D009841:Oligonucleotides; C007673:6-ethylguanine; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase