PREVENTION OF 1-(3-DEOXYCYTIDYL),2-(1-DEOXYGUANOSINYL)ETHANE CROSS-LINK FORMATION IN DNA BY RAT-LIVER O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE

Abstract

Previous studies have shown that the haloethylnitrosoureas introduce the cross-link 1-(3-deoxycytidyl), 2-(1-deoxyguanosinyl)ethane into DNA. This structure is evidently formed by the following sequence of events: an initial attack of a haloethyl group on the O6 position of guanine, formation of the reactive intermediate, 1,O6-ethanoguanine, and reaction of this intermediate with deoxycytidine in the opposite DNA strand. To investigate the role of O6-alkylguanine-DNA alkyltransferase in preventing the formation of this cross-link, a DNA substrate containing O6-fluoroethylguanine has been prepared by reacting DNA with N-2-fluoroethyl-N''-cyclohexyl-N-nitrosourea. The O6-fluoroethylguanine content of this substrate decreases when it is incubated at 37.degree.C and pH 7.8 in the absence of repair factors because of the chemical instability of O6-fluoroethylguanine; however, this loss is accelerated by the addition of rat liver O6-alkylguanine-DNA alkyltransferase, indicating that this repair factor recognizes and repairs O6-fluoroethylguanine in DNA; furthermore, by using [chloroethyl-14C]N-chloroethyl-N''-cyclohexyl-N-nitrosourea, it can be shown directly that the addition of rat liver O6-alkylguanine-DNA, alkyltransferase prevents 1-(3-deoxycytidyl),2-(1-deoxyguanosinyl)ethane formation. These studies link the presence of repair activity to the prevention of a specific cytotoxic lesion in DNA.