Kinetic characterization of a bacteriophage T4 antimutator DNA polymerase

Ping Wu, Nancy Nossal, Stephen Benkovic

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)14748-14755
Number of pages8
JournalBiochemistry
Volume37
Issue number42
DOIs
StatePublished - Oct 20 1998

All Science Journal Classification (ASJC) codes

  • Biochemistry

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