title: Development and validation of a new assay for O6-alkylguanine-DNA-alkyltransferase based on the use of an oligonucleotide substrate, and its application to the measurement of DNA repair activity in extracts of biopsy samples of human urinary bladder mucosa.
abstract: A simplified and highly sensitive assay for the determination of O6-alkylguanine-DNA-alkyltransferase has been developed and validated by the analysis of extracts of human urinary bladder mucosa. The new assay involves the use of a synthetic dodecanucleotide containing a single O6-methylguanine residue as substrate for the enzyme. This substrate is 5'-end-labelled with [35S]PO3 and separation of repaired and unrepaired oligonucleotide is achived by immuno-precipitation with polyclonal antibodies specific for O6-methyldeoxyguanosine. Kinetic analysis of the repair of the oligonucleotide by extracts of Escherichia coli and rat liver showed that the reaction is first-order in substrate and enzyme and gave the molecular rate constants 7.5 x 10(6) mol-1 1-1 sec-1 and 8.0 x 10(6) mol-1 1-1 sec-1, respectively. The rate constants for the repair of the corresponding O6-ethylguanine-containing oligonucleotide were 3.0 x 10(5) mol-1 l-1 sec-1 and 3.6 x 10(6) mol-1 l-1 sec-1, respectively. Analysis of extracts of 48 samples of normal or neoplastic human urinary bladder mucosa obtained by transurethral biopsy or at surgery, by the new method or by a method involving use of [3H]-methylated DNA as substrate and HPLC, indicated excellent agreement between the two methods. The mean AGT content of normal urinary bladder mucosa obtained from individuals without diagnosed bladder cancer was 0.181 +/- 0.081 (mean +/- SD) fmol/microgram protein, that of neoplastic samples 0.323 +/- 0.177 fmol/microgram protein and that of normal tissue obtained from patients with bladder cancer 0.183 +/- 0.068 fmol/microgram protein. The new method is convenient, rapid and extremely sensitive (it can readily measure femtomole quantities of enzyme) and should prove useful for studies of the biochemical epidemiology of DNA repair.
authors.affiliation: Chemical Carcinogenesis Programme, National Hellenic Research Foundation, Athens, Greece.
authors.forename: V L
authors.initials: VL
authors.lastname: Souliotis
authors.forename: A
authors.initials: A
authors.lastname: Giannopoulos
authors.forename: I
authors.initials: I
authors.lastname: Koufakis
authors.forename: S
authors.initials: S
authors.lastname: Kaila
authors.forename: C
authors.initials: C
authors.lastname: Dimopoulos
authors.forename: S A
authors.initials: SA
authors.lastname: Kyrtopoulos
mesh_terms: D000818:Animals; D001706:Biopsy; D004260:DNA Repair; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008780:Methyltransferases / Q000032:analysis* / Q000378:metabolism; D009092:Mucous Membrane / Q000166:cytology / Q000201:enzymology / Q000473:pathology; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009841:Oligonucleotides; D051381:Rats; D012016:Reference Values; D013379:Substrate Specificity; D001743:Urinary Bladder / Q000166:cytology / Q000201:enzymology* / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000201:enzymology* / Q000473:pathology
chemical_list: D009841:Oligonucleotides; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase