Abstract
Synthesis of the leading DNA strand requires the coordinated activity of DNA polymerase and DNA helicase, whereas synthesis of the lagging strand involves interactions of these proteins with DNA primase. We present the first structural model of a bacteriophage T7 DNA helicase-DNA polymerase complex using a combination of small angle x-ray scattering, single-molecule, and biochemical methods. We propose that the protein-protein interface stabilizing the leading strand synthesis involves two distinct interactions: a stable binding of the helicase to the palm domain of the polymerase and an electrostatic binding of the carboxyl-terminal tail of the helicase to a basic patch on the polymerase. DNA primase facilitates binding of DNA helicase to ssDNA and contributes to formation of the DNA helicase-DNA polymerase complex by stabilizing DNA helicase.
Highlights
Interactions of DNA polymerase and DNA helicase are crucial in DNA synthesis
Terminating the primer with a ddNMP and providing the dNTP specified by the template yields a gp5/trx-primer/template complex that mimics ongoing leading strand synthesis, and its structure was solved to a 2.2 Å resolution [6]. gp4 and gp5/trx form a stable complex under these conditions [8]
Gp5/trx and gp4 make numerous interactions. gp5/trx joins the replisome by binding to the C-terminal acidic tail of gp4 via its basic loading patch (Fig. 7A)
Summary
Interactions of DNA polymerase and DNA helicase are crucial in DNA synthesis. Results: Two distinct interactions are involved in formation of the DNA polymerase/DNA helicase complex. Conclusion: The multiple interactions between DNA polymerase and DNA helicase account for the high processivity of leading strand synthesis. Synthesis of the leading DNA strand requires the coordinated activity of DNA polymerase and DNA helicase, whereas synthesis of the lagging strand involves interactions of these proteins with DNA primase. We propose that the proteinprotein interface stabilizing the leading strand synthesis involves two distinct interactions: a stable binding of the helicase to the palm domain of the polymerase and an electrostatic binding of the carboxyl-terminal tail of the helicase to a basic patch on the polymerase. Coordination of leading and lagging strand DNA synthesis, the formation and resolution of a replication loop, and the exchange of DNA polymerases implies that the four proteins interact with one another. DNA Polymerase-DNA Helicase Interaction kb on ssDNA but a processivity of Ͼ17 kb during leading strand synthesis when coupled to gp4 [12]. We dissect the interactions that stabilize the complex of gp and gp5/trx during leading strand synthesis
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