DNA Replication Fidelity with 8-Oxodeoxyguanosine Triphosphate

Oxidative metabolism is known to generate mutagenic compounds within cells, among which is 8-oxodeoxyguanosine. Here the mutagenic potential of the triphosphate form of this base analog (8- O-dGTP) is investigated during replication in vitro of the lacZ α-complementation sequence in M13mp2 DNA. Adding 8-O-dGTP at equimolar concentration with the normal dNTPs to polymerization reactions decreases the fidelity of DNA synthesis by exonuclease-deficient Klenow, T4, and Thermus thermophilus DNA polymerases. Sequence analysis of mutants suggests that 8-O-dGMP is misincorporated opposite template adenines, yielding A ⊒ C transversions. The degree of polymerase selectivity against this error is enzyme-dependent, with rates varying by >25-fold. To determine if the A·8-O-dGMP mispair is proofread, a direct comparison of the fidelity of proofreading-proficient and proofreading-deficient Klenow and T4 DNA polymerases was made. Although the exonuclease activity of Klenow polymerase did not substantially reduce overall misincorporation of 8-O-dGMP, misincorporation was lower for the proofreading-proficient T4 enzyme as compared to its proofreading-deficient derivative. These data suggest that the A·8-O-dGMP mispair can be proofread. The mutagenic potential of 8-O-dGTP with eukaryotic systems was also examined. Misincorporation of 8-O-dGTP opposite adenine was observed during SV40 origin-dependent replication of double-stranded DNA in HeLa cell extracts. When present during replication at a concentration equal to the four normal dNTPs, 8-O-dGTP was at least 13-fold more mutagenic for A·T → C·G transversions than was a 100-fold excess of normal dGTP. These data suggest that 8-O-dGTP could be highly mutagenic during nuclear genomic replication in eukaryotes, with a specificity similar to that in Escherichia coli. DNA polymerase γ, the replicative polymerase for mitochondrial DNA, also readily misincorporated 8-O- dGMP opposite adenine despite the presence of a highly active proofreading exonuclease activity. Given the amount of oxidative metabolism occurring in mitochondria, this result has implications for the stability of the mitochondrial genome and for the origin of degenerative diseases resulting from mitochondrial mutations.