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.

Abstract:

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.

Polymerases:

Topics:

Mutational Analysis, Historical Protein Properties (MW, pI, ...), Nucleotide Incorporation, Exonuclease Activity, Source / Purification

One line summary:

T4 A737V is found to arrest much more frequently than its wild type variant during DNA synthesis; exonuclease activity is indistinguishable from its wild type on single-stranded DNA, while exonuclease activity on duplex DNA is more frequent.

Note:

Need to add pol: T4 A737V/L771F (double mutant)

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 Spacciapoli P1994 3-5' Exonuclease (proofreading) Yes
T4 Spacciapoli P1994 Cloned or native Cloned in E. coli
T4 Spacciapoli P1994 Full length or truncated Full length
T4 Spacciapoli P1994 Specific Activity 3039 units/mg
T4 A737V Spacciapoli P1994 3-5' Exonuclease (proofreading) Yes
T4 A737V Spacciapoli P1994 Cloned or native Cloned in E. coli
T4 A737V Spacciapoli P1994 Full length or truncated Full length
T4 A737V Spacciapoli P1994 Processivity 18bp
T4 A737V Spacciapoli P1994 Specific Activity 741 units/mg
T4 L771F Spacciapoli P1994 3-5' Exonuclease (proofreading) Yes
T4 L771F Spacciapoli P1994 Cloned or native Cloned in E. coli
T4 L771F Spacciapoli P1994 Full length or truncated Full length
T4 L771F Spacciapoli P1994 Specific Activity 2343 Other

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