Format

Send to

Choose Destination
Biochemistry. 1998 Oct 20;37(42):14748-55.

Kinetic characterization of a bacteriophage T4 antimutator DNA polymerase.

Author information

1
Department of Chemistry, The Pennsylvania State University, University Park 16802, USA.

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.

PMID:
9778349
DOI:
10.1021/bi980835a
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center