Kinetic characterization of a bacteriophage T4 antimutator DNA polymerase.
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.
Wu
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Department of Chemistry, The Pennsylvania State University, University Park 16802, USA.
Nossal
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Benkovic
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SJ
D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D017354:Point Mutation; D013379:Substrate Specificity; D017186:Titrimetry
D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases