▪ Abstract  DNA replication fidelity is a key determinant of genome stability and is central to the evolution of species and to the origins of human diseases. Here we review our current understanding of replication fidelity, with emphasis on structural and biochemical studies of DNA polymerases that provide new insights into the importance of hydrogen bonding, base pair geometry, and substrate-induced conformational changes to fidelity. These studies also reveal polymerase interactions with the DNA minor groove at and upstream of the active site that influence nucleotide selectivity, the efficiency of exonucleolytic proofreading, and the rate of forming errors via strand misalignments. We highlight common features that are relevant to the fidelity of any DNA synthesis reaction, and consider why fidelity varies depending on the enzymes, the error, and the local sequence environment.

A study was conducted to determine the fidelity of DNA synthesis catalyzed in vitro by the reverse transcriptase from a human immunodeficiency virus type 1 (HIV-1). Like other retroviral reverse transcriptases, the HIV-1 enzyme does not correct errors by exonucleolytic proofreading. Measurements with M13mp2-based fidelity assays indicated that the HIV-1 enzyme, isolated either from virus particles or from Escherichia coli cells infected with a plasmid expressing the cloned gene, was exceptionally inaccurate, having an average error rate per detectable nucleotide incorporated of 1/1700. It was, in fact, the least accurate reverse transcriptase described to date, one-tenth as accurate as the polymerases isolated from avian myeloblastosis or murine leukemia viruses, which have average error rates of approximately 1/17,000 and approximately 1/30,000, respectively. DNA sequence analyses of mutations generated by HIV-1 polymerase showed that base substitution, addition, and deletion errors were all produced. Certain template positions were mutational hotspots where the error rate could be as high as 1 per 70 polymerized nucleotides. The data are consistent with the notion that the exceptional diversity of the HIV-1 genome results from error-prone reverse transcription.

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Three Pol X family members have been linked to nonhomologous end joining (NHEJ) in mammals. Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates, but the roles of the template-dependent polymerases mu and lambda in NHEJ remain unclear. We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ. However, only pol mu promotes accuracy during immunoglobulin kappa recombination. This distinctive in vivo role correlates with the TdT-like ability of pol mu, but not pol lambda, to act when primer termini lack complementary bases in the template strand. However, unlike TdT, synthesis by pol mu in this context is primarily instructed by a template from another DNA molecule. This apparent gradient of template dependence is largely attributable to a small structural element that is present but different in all three polymerases.

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