Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase

Andrew F Gardner,Catherine M Joyce,William E Jack

doi:10.1074/jbc.m308286200

Andrew F Gardner, Catherine M Joyce + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m308286200

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Abstract

Comparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (Vent(A488L) DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases.

Highlights

All free living organisms encode several DNA polymerases that are jointly responsible for the replication and maintenance of their genomes, thereby ensuring accurate transmission of genetic information [1,2,3]
Analysis of dNTP Incorporation by Vent DNA Polymerase— Previous studies with Family A DNA polymerases have shown that the steady-state rate-limiting step for addition of a single correctly paired dNTP follows phosphodiester bond formation [8, 13, 46, 48]
Incorporation of dCTP by Vent DNA polymerase displayed a burst pattern similar to those seen with RB69 and AmpliTaq-CS DNA polymerases, with a rapid burst followed by slow steady-state turnover (Fig. 3A and Table I)

Summary

EXPERIMENTAL PROCEDURES

Nucleotides, Nucleotide Analogs, DNA Substrate, and Enzymes—All DNA polymerases used in this study are 3Ј 3 5Ј exonuclease-deficient as a result of mutation of catalytic aspartic and glutamic acids to alanine in the exonuclease active site [31, 32, 45]. Rapid quench reactions were carried out as described below with 50 nM FAM-duplex DNA; 10 or 20 nM Vent or VentA488L DNA polymerase; and 0.20 mM dCTP, ddCTP, ddCTP␣S, CTP, or acyCTP (final concentrations after mixing) in 1ϫ ThermoPol buffer (10 mM KCl, 20 mM Tris-HCl (pH 8.8 at 25 °C), 10 mM (NH4)2SO4, 2 mM MgSO4, and 0.1% Triton X-100). Measurement of DNA Polymerase Pre-steady-state Kinetic Parameters—Single turnover nucleotide incorporation reactions were initiated by mixing Vent or VentA488L DNA polymerase (0.10 ␮M) and FAMduplex DNA (0.050 ␮M) in 1ϫ ThermoPol buffer together with an equal volume of nucleotides or nucleotide analogs in 1ϫ ThermoPol buffer. The firstorder rate constant for polymerase-catalyzed addition at each nucleotide concentration was calculated from a plot of ln[substrate] versus time. KD(PPi) and kpyro were derived using fitting protocols analogous to those described above for nucleotide addition

RESULTS

60 Ϯ 40 45 Ϯ 9 230 Ϯ 40 50 Ϯ 7

DISCUSSION

70 Ϯ 7 120 Ϯ 40 1100 Ϯ 100