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PLoS One. 2013 May 16;8(5):e63489. doi: 10.1371/journal.pone.0063489. Print 2013.

Kinetic characterization of exonuclease-deficient Staphylococcus aureus PolC, a C-family replicative DNA polymerase.

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  • 1Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.

Abstract

PolC is the C-family replicative polymerase in low G+C content Gram-positive bacteria. To date several structures of C-family polymerases have been reported, including a high resolution crystal structure of a ternary complex of PolC with DNA and incoming deoxynucleoside triphosphate (dNTP). However, kinetic information needed to understand the enzymatic mechanism of C-family polymerases is limited. For this study we have performed a detailed steady-state and pre-steady-state kinetic characterization of correct dNTP incorporation by PolC from the Gram-positive pathogen Staphylococcus aureus, using a construct lacking both the non-conserved N-terminal domain and the 3'-5' exonuclease domain (Sau-PolC-ΔNΔExo). We find that Sau-PolC-ΔNΔExo has a very fast catalytic rate (k(pol) 330 s(-1)) but also dissociates from DNA rapidly (k(off) ∼150 s(-1)), which explains the low processivity of PolC in the absence of sliding clamp processivity factor. Although Sau-PolC-ΔNΔExo follows the overall enzymatic pathway defined for other polymerases, some significant differences exist. The most striking feature is that the nucleotidyl transfer reaction for Sau-PolC-ΔNΔExo is reversible and is in equilibrium with dNTP binding. Simulation of the reaction pathway suggests that rate of pyrophosphate release, or a conformational change required for pyrophosphate release, is much slower than rate of bond formation. The significance of these findings is discussed in the context of previous data showing that binding of the β-clamp processivity factor stimulates the intrinsic nucleotide incorporation rate of the C-family polymerases, in addition to increasing processivity.

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