A single mutation in bacteriophage T4 DNA polymerase (A737V, tsL141) decreases its processivity as a polymerase and increases its processivity as a 3'-->5' exonuclease.
The bacteriophage T4 DNA polymerase mutant A737V (tsL141 and tsCB120) was originally characterized as temperature-sensitive for DNA replication and an antimutator for transition mutations. Its antimutator phenotype is suppressed by the L771F mutation (Reha-Krantz, L. J., Stocki, S., Nonay, R., and Maughan, C. (1989) J. Cell. Biochem. 13D, 140). We find that the A737V polymerase arrests much more frequently than the wild type when polymerizing on primed single-stranded DNA templates. Although the 3'-->5' exonuclease of the mutant is indistinguishable from the wild type on single-stranded DNA, it is more active than the wild type on duplex DNA. In a single encounter with the primer, the wild type polymerase can incorporate more than 50 nucleotides. The processivity of the A737V polymerase is less than the wild type as a polymerase, but is greater than the wild type as an exonuclease. The L771F polymerase resembles the wild type in each of these properties, while the double mutant (A737V, L771F) is intermediate between the two single mutants. Kinetic studies of wild type T4 DNA polymerase (Capson, T. L., Peliska, J. A., Kaboord, B. F., Frey, M. W., Lively, C., Dahlberg, M., and Benkovic, S. J. (1992) Biochemistry 31, 10984-10994) suggest that DNA binds first to the polumerase active site, before adopting a configuration in which it can be hydrolyzed by the exonuclease. Within this framework, our studies suggest that DNA moves more readily from the polymerase- to the exonuclease-competent configuration on the A737V mutant polymerase, and that this movement is decreased by the compensating L771F mutation.
Spacciapoli
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Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
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D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D013698:Templates, Genetic
D009838:Oligodeoxyribonucleotides; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V