Kinetic characterization of a bacteriophage T4 antimutator DNA polymerase

Biochemistry. 1998 Oct 20;37(42):14748-55. doi: 10.1021/bi980835a.


Fidelity of DNA replication by bacteriophage T4 DNA polymerase is achieved in a multiplicative process: base selection by its polymerase activity and removal of misincorporated nucleotides by its exonuclease activity. The wild-type polymerase is capable of maintaining a balance between the two activities so that DNA replication fidelity is maximized without excessive waste of nucleotides. Antimutator enzymes exhibit a higher DNA replication fidelity than the wild-type enzyme, at the cost of increased nucleotide turnover. The antimutator A737V polymerase has been characterized kinetically using pre-steady-state and steady-state methods to provide a kinetic sequence which defines the effect of the mutation on the discrete steps controlling DNA replication fidelity. Comparison of this sequence to that of the wild type [Capson, L. T., Peliska, J. A., Kaboord, B. F., Frey, M. W., Lively, C., Dahlberg, M., and Benkovic, S. J. (1992) Biochemistry 31, 10984-10994] revealed that A737V polymerase differs in two ways. The rates at which DNA is transferred between the exonuclease and polymerase sites are reduced approximately 7-fold for a duplex DNA containing a mismatched 3'-terminus, and the partitioning of the mismatched duplex between the polymerase and exonuclease sites is 1:2 versus 4:1 for the wild-type enzyme. The exonuclease activity of A737V relative to the wild-type enzyme is unchanged on single-stranded DNA. However, the difference in partitioning the duplex DNA between the exonuclease and polymerase active sites results in an enhanced exonuclease activity for the antimutator enzyme.

Publication types

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

MeSH terms

  • Bacteriophage T4 / enzymology*
  • Bacteriophage T4 / genetics*
  • Bacteriophage T4 / metabolism
  • Binding Sites / genetics
  • DNA / metabolism
  • DNA, Single-Stranded / metabolism
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism
  • Exodeoxyribonucleases / metabolism
  • Kinetics
  • Models, Chemical
  • Nucleic Acid Heteroduplexes / chemistry
  • Point Mutation*
  • Substrate Specificity
  • Titrimetry


  • DNA, Single-Stranded
  • Nucleic Acid Heteroduplexes
  • DNA
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases