The fidelity of the human leading and lagging strand DNA replication apparatus with 8-oxodeoxyguanosine triphosphate

A product of oxidatlve metabolism, 8-oxodeoxyguanosine triphosphate (8-O-dGTP), readily pairs with adenlne during DNA replication, ultimately causing A.T→C.G transverslons. This study utilized 8-O-dGTP as a probe to examine the fidelity of the leading and lagging strand replication apparatus In extracts of HeLa cells. Simian virus (SV) 40 T antigen-dependent DNA replication reactions were performed with two M13mp2 vectors with the SV40 origin located on opposite sides of the lacZα sequence used to score replication errors. The presence of 8-O-dGTP at equimolar concentration with each of the 4 normal dNTPs resulted In a ≫ 46-fold increase in error rate for A.T→C.G transverslons over that observed in the absence of 8-O-dGTP. A similar average error rate was observed on the (+) and (-) strands In both vectors, suggesting that the fidelity of replication by leading and lagging strand replication proteins Is similar for the dA-8-O-dGMP mispalr. Replication fidelity In the presence of 8-O-dGTP was reduced on both strands when an inhibitor of exonucleolytlc proofreading (dGMP) was added to the reaction. These data suggest that the majority of dA-8-O-dGMP mlspalrs are proofread by both leading and lagging strand replication proteins.