DNA replication fidelity with 8-oxodeoxyguanosine triphosphate

Biochemistry. 1994 Apr 19;33(15):4695-701. doi: 10.1021/bi00181a029.

Abstract

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 alpha-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriophage T4 / enzymology
  • Base Composition
  • DNA / biosynthesis*
  • DNA Polymerase I
  • DNA Replication*
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyguanine Nucleotides / metabolism*
  • Escherichia coli / genetics
  • Glycosylation
  • Mutagenesis
  • Thermus thermophilus / enzymology
  • Transfection

Substances

  • Deoxyguanine Nucleotides
  • 8-oxodeoxyguanosine triphosphate
  • DNA
  • DNA Polymerase I
  • DNA-Directed DNA Polymerase