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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)

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Sphingosine is a potent inhibitor of a mammalian DNA primase in vitro (Simbulan et al., Biochemistry 33, 9007‐9012, 1994). Here we measured the inhibition of DNA primase in vitro by 9 sphingosine‐analogues with respect to RNA primer synthesis and DNA primase‐dependent DNA synthesis, and their potencies of inhibition in vitro were compared with their in vivo effects on human leukemic cells. Sphingosine, phytosphingosine and N, N‐dimethylsphingosine strongly inhibited the activity of purified calf thymus DNA primase, and also inhibited the growth of human leukemic cell line HL‐60, exerting strong cytotoxicity. Dihydrosphingosine and cis‐sphingosine, which showed more subtle inhibition of DNA primase in vitro, moderately inhibited the cell growth in vivo and caused cell death. In contrast, N‐acyl‐, N‐octyl‐, and N‐acetylsphingosine (ceramides) showing little inhibition of DNA primase suppressed cell growth only slightly. HL 60 cell was arrested at Go/G1 phase by exogenously added sphingosine. From these results, it is suggested that DNA primase is one of targets of sphingosine, an effector molecule in apoptosis.