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Nucleic Acids Research, 1994, Vol. 22, No. 25 5658-5664
© 1994

Articles

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

Dana T. Minnick, Youri I. Pavlov1 and Thomas A. Kunkel*

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences Research Triangle Park, NC 27709, USA 1 Department of Genetics, St Petersburg University, Universitetskaya emb 7/9, St Petersburg 199034, Russia

*To whom correspondence should be addressed

Received August 25, 1994. Revised November 12, 1994. Accepted November 12, 1994.

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 lacZ{alpha} 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.


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