The effect of genotoxic compounds on the fidelity of DNA synthesis

• Main Author: Power, Christine Anna Grek
• Published: University of Surrey 1983
• Subjects: 572; Biochemistry
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.344402

The fidelity with which DNA polymerases replicate the synthetic polynucleotide polydA. oligodT, can be determined in vitro by measuring the ratio of incorporation of non-complementary nucleotides and complementary nucleotides into polymeric material. In order to study the effect of in vivo or in vitro administration of various genotoxic agents on the activity and fidelity of DNA polymerases associated with DNA repair, adult rat liver nuclear extracts were used as a source of DNA polymerase activity. Endogenous DNA in nuclear extracts did not interfere with fidelity studies since no DNA polymerase activity was detectable in the absence of polydA. oligodT. Extracts were shown to contain almost exclusively DNA polymerase beta (the putative mammalian DNA repair enzyme) by the use of differential inhibitors of DNA polymerases. However, DNA polymerase beta fidelity in nuclear extracts was almost 10-fold lower than the fidelity measured for some purified DNA polymerases. In vitro modification of DNA polymerase beta activity in nuclear extracts by increasing concentrations of the alkylating agents, methyl methane sulphonate (1-400mM) beta-propiolactone (1-100mM) and N-methyl-N-nitro-N-nitrosoguanidine (1-20mM) resulted in a gradual inactivation of the enzyme. A similar effect was observed upon treatment of the enzyme with anti(+/-)BPDE. The addition of these agents to nuclear extracts appeared to enhance the fidelity of DNA polymerase beta activity. In vivo administration of methyl methane sulphonate also resulted in an inhibition of DNA polymerase beta activity in nuclear extracts but no change in fidelity was observed. In vivo administration of a series of N,-N-dialkylnitrosamines generally resulted in an increase in the total DNA polymerase beta activity in rat liver nuclei. Dimethylnitrosamine and diethylnitrosamine appeared to induce an error-prone DNA polymerase activity whereas longer alkyl derivatives, dipropylnitrosamine and dibutylnitrosamine did not. Similarly in vivo administration of 2 toxic doses of benzo(a)pyrene to rats 48h and 24h before killing resulted in an increase in DNA polymerase B activity in rat liver nuclei. A single dose of benzo(a)pyrene depressed total DNA polymerase beta activity in nuclei for up to 14 days. The benzo(a)pyrene treatments used in these studies failed to induce an error-prone DNA polymerase activity in rat liver nuclei.