Molecular Mechanisms of the Functional Coupling of the Helicase (gp41) and Polymerase (gp43) of Bacteriophage T4 within the DNA Replication Fork

• Published in: Biochemistry (Easton) 2001-04, Vol.40 (14), p.4459-4477
• Main Authors: Delagoutte, Emmanuelle, von Hippel, Peter H
• Format: Article
• Language: English
• Subjects: Bacteriophage T4 - enzymology; Dimerization; DNA Helicases - chemistry; DNA Helicases - metabolism; DNA Helicases - physiology; DNA Replication; DNA, Viral - biosynthesis; DNA, Viral - chemistry; DNA-Directed DNA Polymerase - chemistry; DNA-Directed DNA Polymerase - metabolism; DNA-Directed DNA Polymerase - physiology; Protein Processing, Post-Translational; Salts; Ultracentrifugation; Viral Proteins - chemistry; Viral Proteins - metabolism; Viral Proteins - physiology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001306l

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 ∼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 ∼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.