The A, B, C, D’s of replicative DNA polymerase fidelity: utilizing high-throughput single-molecule sequencing to understand the molecular basis for DNA polymerase accuracy

DNA polymerases (DNAPs) are indispensable enzymes that play central roles in biology, by replicating and repairing genetic material, as well as in biotechnology, by fueling such innovations as polymerase chain reaction (PCR), cloning, and DNA sequencing. Replicative DNAPs possess dual catalytic activities that work together for high accuracy replication: a selective DNA-dependent DNA polymerase activity for synthesizing DNA and a proofreading exonuclease activity for removing misincorporated nucleotides. Despite their precision, DNA polymerases occasionally make errors, and understanding the mechanisms behind these mistakes is essential to fully leverage these enzymes. Indeed, measuring DNA polymerase fidelity not only reveals the basis of their accuracy, but also enables rational modulation of their fidelity. Here we employ a highly accurate Pacific Biosciences sequencing workflow that leverages long-read, non-PCR-based technology to measure DNAP error rates and profiles. By measuring the fidelity of the four primary replicative DNA polymerase families, A, B, C, and D, measurements uncovered remarkably diverse family specific error profiles. Factors that influence DNAP fidelity, such as deoxynucleoside triphosphate ratios, replication components, and exonuclease and polymerase active site mutations, are further explored. This work deepens our understanding of DNA replication, the mechanisms that underly DNA polymerase fidelity, and informs development of advanced DNA polymerase-based tools for biotechnology.