Steady-state kinetic characterization of RB69 DNA polymerase mutants that affect dNTP incorporation

Biochemistry. 1999 Jun 22;38(25):8094-101. doi: 10.1021/bi990653w.


The function of six highly conserved residues (Arg482, Lys483, Lys486, Lys560, Asn564, and Tyr567) in the fingers domain of bacteriophage RB69 DNA polymerase (RB69 gp43) were analyzed by kinetic studies with mutants in which each of these residues was replaced with Ala. Our results suggest that Arg482, Lys486, Lys560, and Asn564 contact the incoming dNTP during the nucleotidyl transfer reaction as judged by variations in apparent Km and kcat values for dNTP incorporation by these mutants compared to those for the exonuclease deficient parental polymerase under steady-state conditions. On the basis of our studies, as well as on the basis of the crystal structure of RB69 gp43, we propose that a conformational change in the fingers domain, which presumably occurs prior to polymerization, brings the side chains of Arg482, Lys486, Lys560, and Asn564 into the vicinity of the primer-template terminus where they can contact the triphosphate moiety of the incoming dNTP. In particular, on the basis of structural studies reported for the "closed" forms of two other DNA polymerases and from the kinetic studies reported here, we suggest that (i) Lys560 and Asn564 contact the nonbonding oxygens of the alpha and beta phosphates, respectively, and (ii) both Arg482 and Lys486 contact the gamma phosphate oxygens of the incoming dNTP of RB69 gp43 prior to the nucleotidyl transfer reaction. We also found that Ala substitutions at each of these four RB69 gp43 sites could incorporate dGDP as a substrate, although with markedly reduced efficiency compared to that with dGTP. In contrast in the parental exo- background, the K483A and Y567A substituted enzymes could not use dGDP as a substrate for primer extension. These results, taken together, are consistent with the putative roles of the four conserved residues in RB69 gp43 as stated above.

Publication types

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

MeSH terms

  • Alanine / genetics
  • Asparagine / genetics
  • Bacteriophage T4 / enzymology
  • Bacteriophage T4 / genetics
  • DNA-Directed DNA Polymerase / chemical synthesis
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyguanine Nucleotides / metabolism
  • Dinucleoside Phosphates / metabolism*
  • Enzyme Activation / genetics
  • Exodeoxyribonucleases / metabolism
  • Kinetics
  • Lysine / genetics
  • Mutagenesis, Site-Directed
  • Plasmids / chemical synthesis
  • Protein Structure, Secondary
  • Substrate Specificity / genetics
  • Tyrosine / genetics
  • Viral Proteins / chemical synthesis
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism*


  • Deoxyguanine Nucleotides
  • Dinucleoside Phosphates
  • Viral Proteins
  • gene 43 protein, Enterobacteria phage T4
  • Tyrosine
  • Asparagine
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • Lysine
  • Alanine