Pre-steady-state kinetics of RB69 DNA polymerase and its exo domain mutants: effect of pH and thiophosphoryl linkages on 3'-5' exonuclease activity

Biochemistry. 2004 Apr 6;43(13):3853-61. doi: 10.1021/bi0302292.


DNA polymerases from the A and B families with 3'-5' exonucleolytic activity have exonuclease domains with similar three-dimensional structures that require two divalent metal ions for catalysis. B family DNA polymerases that are part of a replicase generally have a more potent 3'-5' exonuclease (exo) activity than A family DNA polymerases that mainly function in DNA repair. To investigate the basis for these differences, we determined pH-activity profiles for the exonuclease reactions of T4, RB69, and phi29 DNA polymerases as representatives of B family replicative DNA polymerases and the Klenow fragment (KF) as an example of a repair DNA polymerase in the A family. We performed exo assays under single-turnover conditions and found that excision rates exhibited by the B family DNA polymerases were essentially independent of pH between pH 6.5 and 8.5, whereas the exo activity of KF increased 10-fold for each unit increase in pH. Three exo domain mutants of RB69 polymerase had much lower exo activities than the wild-type enzyme and exhibited pH-activity profiles similar to that of KF. On the basis of pH versus activity data and elemental effects obtained using short double-stranded DNA substrates terminating in phosphorothioate linkages, we suggest that the rate of the chemical step is reduced to the point where it becomes limiting with RB69 pol mutants K302A, Y323F, and E116A, in contrast to the wild-type enzyme where chemistry is faster than the rate-determining step that precedes it.

Publication types

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

MeSH terms

  • Alanine / genetics
  • Amino Acid Substitution / genetics
  • Bacteriophage T4 / enzymology
  • Bacteriophage T4 / genetics
  • Base Pair Mismatch / genetics
  • DNA Polymerase I / chemistry
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics*
  • Enzyme Activation / genetics
  • Exodeoxyribonucleases / chemistry*
  • Exodeoxyribonucleases / genetics*
  • Glutamine / genetics
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mutagenesis, Site-Directed*
  • Phosphates / chemistry*
  • Phosphorylation
  • Protein Structure, Tertiary / genetics
  • RNA Editing / genetics
  • Substrate Specificity / genetics
  • T-Phages / enzymology*
  • T-Phages / genetics*
  • Thionucleotides / chemistry
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics*


  • Phosphates
  • Thionucleotides
  • Viral Proteins
  • Glutamine
  • DNA Polymerase I
  • DNA-Directed DNA Polymerase
  • bacteriophage RB69 DNA polymerase
  • Exodeoxyribonucleases
  • Alanine
  • thiophosphoric acid