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  • How Does a High-Fidelity Polymerase Ensure that the Correct Base is Inserted?

    Return to Q5® High-Fidelity DNA Polymerase Products

    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.

    References

    1. Johnson, K. A. (2010) Biochimica et Biophysica Acta, 1804, 1041–1048. PMID: 20079883
    2. Joyce, C. M., & Benkovic, S. J. (2004) Biochemistry, 43, 14317–14324. PMID: 15533035