Pre-steady-state kinetic studies of the fidelity of Sulfolobus solfataricus P2 DNA polymerase IV.

Biochemistry (2004), Volume 43, Page 2106

Abstract:

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a thermostable archaeal enzyme and a member of the error-prone and lesion-bypass Y-family. In this paper, for the first time, the fidelity of a Y-family polymerase, Dpo4, was determined using pre-steady-state kinetic analysis of the incorporation of a single nucleotide into an undamaged DNA substrate 21/41-mer at 37 degrees C. We assessed single-turnover (with Dpo4 in molar excess over DNA) saturation kinetics for all 16 possible nucleotide incorporations. The fidelity of Dpo4 was estimated to be in the range of 10(-3)-10(-4). Interestingly, the ground-state binding affinity of correct nucleotides (70-230 microM) is 10-50-fold weaker than those of replicative DNA polymerases. Such a low affinity is consistent with the lack of interactions between Dpo4 and the bound nucleotides as revealed in the crystal structure of Dpo4, DNA, and a matched nucleotide. The affinity of incorrect nucleotides for Dpo4 is approximately 2-10-fold weaker than that of correct nucleotides. Intriguingly, the mismatched dCTP has an affinity similar to that of the matched nucleotides when it is incorporated against a pyrimidine template base flanked by a 5'-template guanine. The incoming dCTP likely skips the first available template base and base pairs with the 5'-template guanine, as observed in the crystal structure of Dpo4, DNA, and a mismatched nucleotide. The mismatch incorporation rates, regardless of the 5'-template base, were approximately 2-3 orders of magnitude slower than the incorporation rates for matched nucleotides, which is the predominant contribution to the fidelity of Dpo4.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Other Enzymatic Activities, Kinetic Parameters, Structure and Structure/Function, Fidelity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Dpo4 Fiala KA2004 Cloned or native Cloned in E. coli
Dpo4 Fiala KA2004 Tagged Yes
Dpo4 Fiala KA2004 Tag Name C-terminal 6-His
Dpo4 Fiala KA2004 Overall Error Rate 0.00015 errors/bp
Dpo4 Fiala KA2004 Overall Error Rate 0.0032 errors/bp
Dpo4 Fiala KA2004 Full length or truncated Full length
Dpo4 Fiala KA2004 Vmax 3.8 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
Eco Pol I Fiala KA2004 Overall Error Rate 0.00017 errors/bp
Eco Pol I Fiala KA2004 Overall Error Rate 0.00053 errors/bp
Human Pol gamma Fiala KA2004 Overall Error Rate 1.8E-06 errors/bp
Human Pol gamma Fiala KA2004 Overall Error Rate 0.00029 errors/bp
T7 Fiala KA2004 Overall Error Rate 2.6E-07 errors/bp
T7 Fiala KA2004 Overall Error Rate 6.7E-06 errors/bp

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