Pre-Steady-State Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9{degrees}N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance [DNA and Chromosomes]

July 9th, 2015 by Schermerhorn, K. M., Gardner, A. F.

Family D (polD) DNA polymerases have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, k pol and K d, for correct, incorrect, and ribo- nucleotide incorporation by exonuclease deficient polD. Correct nucleotide incorporation kinetics revealed relatively slow maximal rate of polymerization ( k pol ≈2.5 s-1) and especially tight nucleotide binding (KddNTP ≈1.7 μM), compared to DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed polD prevents the incorporation of incorrect and ribo- nucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3′-5′ exonuclease hydrolysis activity in the presence of Mg2+ and Mn2+. Interestingly, substituting Mn2+ for Mg2+ accelerated hydrolysis rates over 40-fold (kexo ≥110 s-1 versus ≥2.5 s-1). Preference for Mn2+ over Mg2+ in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests a conserved polymerase kinetic pathway is present in all known DNA polymerase families.
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