Molecular mechanisms of the functional coupling of the helicase (gp41) and polymerase (gp43) of bacteriophage T4 within the DNA replication fork.

Biochemistry (2001), Volume 40, Page 4459

Abstract:

Processive strand-displacement DNA synthesis with the T4 replication system requires functional "coupling" between the DNA polymerase (gp43) and the helicase (gp41). To define the physical basis of this functional coupling, we have used analytical ultracentrifugation to show that gp43 is a monomeric species at physiological protein concentrations and that gp41 and gp43 do not physically interact in the absence of DNA, suggesting that the functional coupling between gp41 and gp43 depends significantly on interactions modulated by the replication fork DNA. Results from strand-displacement DNA synthesis show that a minimal gp41-gp43 replication complex can perform strand-displacement synthesis at approximately 90 nts/s in a solution containing poly(ethylene glycol) to drive helicase loading. In contrast, neither the Klenow fragment of Escherichia coli DNA polymerase I nor the T7 DNA polymerase, both of which are nonprocessive polymerases, can carry out strand-displacement DNA synthesis with gp41, suggesting that the functional helicase-polymerase coupling may require the homologous system. However, we show that a heterologous helicase-polymerase pair can work if the polymerase is processive. Strand-displacement DNA synthesis using the gp41 helicase with the T4 DNA polymerase holoenzyme or the phage T7 DNA polymerase-thioredoxin complex, both of which are processive, proceeds at the rate of approximately 250 nts/s. However, replication fork assembly is less efficient with the heterologous helicase-polymerase pair. Therefore, a processive (homologous or heterologous) "trailing" DNA polymerase is sufficient to improve gp41 processivity and unwinding activity in the elongation stage of the helicase reaction, and specific T4 helicase-polymerase coupling becomes significant only in the assembly (or initiation) stage.

Polymerases:

Topics:

Kinetic Parameters, Accessory Proteins/Complexes, Nucleotide Incorporation

One line summary:

Functional coupling of T4 helicase (gp41) and T4 DNA polymerase

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 D219A Delagoutte E2001 Vmax 230 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
T4 D219A Delagoutte E2001 Vmax 0 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
T4 D219A Delagoutte E2001 Vmax 125 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
T4 D219A Delagoutte E2001 Vmax 55 /second Reaction: Nucleotide incorporation; Substrate: dNTPs

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