Understanding the Fidelity and Specificity of DNA Polymerase I

High-fidelity DNA polymerases employ complex mechanisms to catalyze template-dependent DNA synthesis while quickly discarding mismatches. Atomic-level structural details about short-lived states during nucleotide discrimination are necessary to gain insight into the kinetic checkpoints that contribute to fidelity. We performed microsecond molecular dynamics simulations of DNA polymerase I, large fragment, from Bacillus stearothermophilus (Bacillus fragment, or BF) in complex with a template guanine and a mismatched thymidine triphosphate to observe the early events in the process of selection against a mismatch. Although the nucleobases formed a wobble base pair early, the mismatched pair was blocked from fully entering the active site by a conserved tyrosine, Tyr714, leading to the eventual disruption of the unstable pair. Simulations of the mutant BF at residue 714 reveal that a serine mutation readily accommodates a G-T mismatch, explaining the results of previous studies. Mismatch G-G simulations reproduce previous DNA polymerase crystal structures and further support the importance of Tyr714 in DNA polymerase fidelity. Our molecular dynamics studies of BF provide strong evidence for a multiconformational, stepwise selection mechanism that disfavors unstable mismatches prior to closure. Our free energy calculations indicate a substantial barrier between the closed and ajar states. This suggests that once the ternary complex fully closes, it will likely remain closed, regardless of whether complementary or noncomplementary nucleotides are present in the active site. Dynamic discrimination against mismatches leads to nucleotide dissociation and contributes to DNA replication fidelity in DNA polymerase I.