Interaction of DNA polymerase and DNA helicase within the bacteriophage T4 DNA replication complex. Leading strand synthesis by the T4 DNA polymerase mutant A737V (tsL141) requires the T4 gene 59 helicase assembly protein.

Abstract:

The bacteriophage T4 tsL141 (A737V) mutant in T4 DNA polymerase is temperature-sensitive for DNA replication and an antimutator for some types of mutations. In the accompanying paper (Spacciapoli, P., and Nossal, N. G. (1993) J. Biol. Chem. 269, 438-446), we show that the purified A737V T4 DNA polymerase is less processive than the wild type enzyme as a polymerase, but is more processive as an exonuclease. The bacteriophage T4 multienzyme replication complex reconstituted with the A737V mutant polymerase is defective in both lagging and leading strand synthesis. On lagging strand templates, the A737V polymerase is stimulated by the gene 44/62 and 45 polymerase accessory proteins and the gene 32 DNA binding protein, but is still arrested at pause sites much more frequently than the wild type. In contrast to wild type T4 DNA polymerase, the A737V polymerase does not catalyze leading strand synthesis on a forked duplex template with the polymerase accessory proteins, 32 protein, and the gene 41 protein helicase. The A737V polymerase requires the T4 gene 59 helicase assembly protein, as well as the other proteins, to carry out this reaction. Each of these defects is suppressed by the intragenic L771F mutation that suppresses the antimutator phenotype of the A737V, polymerase in vivo (Reha-Krantz, L. J., Stocki, S., Nonay, R., and Maughan, C. (1989) J. Cell. Biochem. 13D, 140).

Polymerases:

Topics:

Mutational Analysis, Structure and Structure/Function, Accessory Proteins/Complexes, Nucleotide Incorporation

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