High-fidelity DNA polymerases have several checkpoints to protect against making and propagating mistakes while copying DNA.
- High-fidelity polymerases have a significant binding preference for the correct versus the incorrect nucleotide triphosphate during polymerization.
- If an incorrect nucleotide does bind in the polymerase active site, incorporation is slowed due to the sub-optimal architecture of the active site complex. This time increases the opportunity for the incorrect nucleotide to dissociate before incorporation, thereby allowing the process to start again (and for a correct nucleotide triphosphate to bind) (1,2).
- If an incorrect nucleotide is inserted, proof-reading DNA polymerases have an extra line of defense. They can "sense" the perturbation caused by the mispaired bases and move the 3' end of the growing DNA chain into a proofreading 3'→5' exonuclease domain. There, the incorrect nucleotide is removed by the 3'→5' exonuclease activity before the chain is moved back into the polymerase domain, where polymerization can continue with the correct nucleotide.