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

Biochemistry. 2004 Feb 24;43(7):2106-15. doi: 10.1021/bi0357457.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / isolation & purification
  • Base Pair Mismatch
  • Binding Sites
  • Catalysis
  • DNA Polymerase beta / chemistry*
  • DNA Polymerase beta / isolation & purification
  • DNA Replication*
  • DNA, Archaeal / biosynthesis*
  • DNA, Archaeal / chemistry
  • Deoxycytosine Nucleotides / chemistry
  • Enzyme Stability
  • Kinetics
  • Models, Chemical
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Substrate Specificity
  • Sulfolobus / enzymology*
  • Thymine Nucleotides / chemistry

Substances

  • Archaeal Proteins
  • DNA, Archaeal
  • Deoxycytosine Nucleotides
  • Recombinant Proteins
  • Thymine Nucleotides
  • 2'-deoxycytidine 5'-triphosphate
  • DNA Polymerase beta
  • thymidine 5'-triphosphate