# Understanding the Fidelity and Specificity of DNA Polymerase I

## TL;DR

This study explores how DNA polymerase I rejects mismatched nucleotides during DNA replication using molecular simulations.

## Contribution

The study reveals a multiconformational selection mechanism involving Tyr714 that blocks mismatched nucleotides before complex closure.

## Key findings

- A conserved tyrosine residue blocks mismatched nucleotides from entering the active site.
- Free energy calculations show a high barrier between closed and ajar states of the polymerase.
- Simulations support a stepwise discrimination mechanism that enhances replication fidelity.

## Abstract

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.