The Reopening Rate of the Fingers Domain Is a Determinant of Base Selectivity for RB69 DNA Polymerase

Biochemistry. 2009 Mar 17;48(10):2087-98. doi: 10.1021/bi8016284.

Abstract

Two divalent metal ions are required for nucleotide incorporation by DNA polymerases. Here we use the bacteriophage RB69 DNA polymerase (RB69 pol) and the metal ion exchange-inert nucleotide analogue rhodium(III) deoxythymidine triphosphate (Rh.dTTP) to investigate the requirements of metal binding to the "A" site and to the "B" site, independently. We show that while binding of a metal ion to the A site is required for the nucleotidyl transfer reaction to occur, this metal binding is insufficient to initiate the prechemistry enzyme isomerization that has been observed with this polymerase. Moreover, we show that binding of a deoxynucleoside triphosphate (dNTP), in the absence of a catalytic metal ion, is sufficient to induce this conformational change. In this report, we also present several lines of evidence (from pulse-chase, rapid chemical quench-flow, and stopped-flow fluorescence experiments) for the reverse rate of the enzyme isomerization, closed to open, of a DNA polymerase complex. The implications of these data for the fidelity of DNA polymerization by RB69 pol are discussed.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 2-Aminopurine / chemistry
  • Adenine Nucleotides / chemistry
  • Base Pair Mismatch
  • Biocatalysis
  • Calcium / chemistry
  • Catalytic Domain / physiology
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxycytosine Nucleotides / chemistry
  • Kinetics
  • Magnesium / chemistry
  • Models, Molecular
  • Oligodeoxyribonucleotides / chemistry
  • Protein Structure, Tertiary / physiology
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Rhodium / chemistry
  • Spectrometry, Fluorescence
  • Substrate Specificity / physiology
  • Thymine Nucleotides / chemistry
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • Adenine Nucleotides
  • Deoxycytosine Nucleotides
  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • Thymine Nucleotides
  • Viral Proteins
  • 2'-deoxycytidine 5'-triphosphate
  • 2-Aminopurine
  • Rhodium
  • DNA-Directed DNA Polymerase
  • bacteriophage RB69 DNA polymerase
  • Magnesium
  • thymidine 5'-triphosphate
  • Calcium