FIDELITY OF MAMMALIAN DNA-REPLICATION AND REPLICATIVE DNA-POLYMERASES

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ-alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater-than-or-equal-to pol-epsilon > pol-delta > pol-alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase-epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol-epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase-delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol-epsilon. In contrast to pol-epsilon , pol-delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100-mu-M), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase-delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen. DNA sequence analysis of independent mutants generated by each enzyme shows that they all produce single-base substitution and frameshift errors, as well as larger deletions. However, the three polymerases have distinctly different error rates and error specificities, which has implications for their roles in the various stages of DNA replication.