Comparative kinetics of nucleotide analog incorporation by vent DNA polymerase.

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.

Polymerases:

Topics:

Mutational Analysis, Biotech Applications, Kinetic Parameters, Nucleotide Incorporation, Methods, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Vent A488L Gardner AF2004 Incorporation of non-standard nucleotides 10 - 50%
Vent A488L Gardner AF2004 Template lesions Bypasses
Vent A488L Gardner AF2004 Methods Featured Gel Shift (EMSA)
Vent A488L Gardner AF2004 Cloned or native Cloned in E. coli
Vent A488L Gardner AF2004 Tagged No
Vent A488L Gardner AF2004 Full length or truncated Unspecified
Vent A488L Gardner AF2004 Application name Sequencing
Vent A488L Gardner AF2004 Kd 1100uM Reaction: Pyrophosphorolysis; Substrate: DNA template; Technique: Rapid quench ; Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 Kd 77uM Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Rapid quench ; Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 Kd 360uM Reaction: Pyrophosphorolysis; Technique: Rapid quench ; Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 Kd 18uM Reaction: Nucleotide incorporation; Substrate: ddCTP; Technique: Rapid quench ; Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 Kd 24uM Reaction: Nucleotide incorporation; Substrate: CTP analog; Technique: Rapid quench ; Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 kcat 0.47 /second Reaction: Nucleotide incorporation; Substrate: ddCTP; Technique: Rapid quench (burst conditions); Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 kcat 13 /second Technique: Rapid quench (single turnover); Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 kcat 0.63 /second Reaction: Pyrophosphorolysis; Substrate: DNA template; Technique: Rapid quench (single turnover); Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 kcat 56 /second Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Rapid quench (single turnover); Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 kcat 0.7 /second Reaction: Nucleotide incorporation; Substrate: rCTP; Technique: Rapid quench (single turnover); Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 kcat 0.3 /second Reaction: Nucleotide incorporation; Substrate: ddCTP; Technique: Rapid quench (single turnover); Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 kcat 13 /second Reaction: Nucleotide incorporation; Substrate: CTP analog; Technique: Rapid quench (single turnover); Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 kcat 45 /second Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Rapid quench (burst conditions); Experimental conditions: Mg++ (2mM), pH (pH 8.8), Temp (60°C)
Vent A488L Gardner AF2004 Gap Filling Unspecified

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