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.

Direct assay for O6-methylguanine-DNA methyltransferase and comparison of detection methods for the methylated enzyme in polyacrylamide gels and electroblots

“…We describe a direct assay for 06-MT activity in cell and tissue extracts which monitors the transfer of radiolabelled methyl groups from the 06 atoms of guanine in DNA to protein. As similarly found by others using different enzyme assays [9,12,15,17,[30][31][32][33], this transfer at 37°C is rapid with, in the present study, 50% of the reaction against double-stranded substrate occurring within the first I min of incubation. The single-stranded form of this substrate displays a much lower rate of reaction with 06-MT [9,34,35], the human spleen enzyme taking 4 h to reach near-completion ( Fig.…”

“…In an attempt to improve sensitivity, several indirect assays have been described which use 32P-labelled oligodeoxynucleotides containing 06-methylguanine, where conversion of the methylated into the non-methylated substrate was monitored by h.p.l.c. [12,13] or, more recently, by immunoassay [14,15]. Two substantial problems that need to be overcome before oligonucleotide substrates find general suitability for use in the measurement of 06-methylguanine repair are, firstly, that extracts prepared from some tissues (colon, spleen and small intestine have thus far been identified) may cause the rapid degradation of the oligodeoxynucleotide substrate [13].…”

Evolutionary consequences of nonrandom damage and repair of chromatin domains

“…Individuals among humans may display different levels in this enzyme activity (Oesch et al 1987;Souliotis et al 1989), and some deficiencies in this enzyme may increase cancer risk (Montesano 1981). Both the sites of formation and repair of O6-methylguanine are nonrandomly distributed on DNA (Topal et al 1986;Topal 1988).…”

Προσδιορισμος Υποδοχεων Ιντερφερονης Α Στον Καρκινο Της Ουροδοχου Κυστεως Και Στο Φυσιολογικο Ουροθηλιο

“…Η σημαντικότερη προκαρκινική βλάβη την οποία προκαλούν τα καρκινογόνα στο DNA των κυττάρων στόχων, είναι η αλκυλίωση της γουανίνης στη θέση 0 6 καί ο σχηματισμός 0 6 -αλκυλογουανίνης. Την προκαρκινική της ικανότητα η αλκυλιωμένη γουανίνη την οφείλει στην λανθασμένη κωδικοποίηση του DNA που προκαλεί, κατά την διάρκεια του αναδιπλασιασμού των κυττάρων (63).…”