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Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome

Nature Chemical Biology volume 5, pages 904–912 (2009)Cite this article

Abstract

The unwinding and priming activities of the bacteriophage T4 primosome, which consists of a hexameric helicase (gp41) translocating 5′ to 3′ and an oligomeric primase (gp61) synthesizing primers 5′ to 3′, have been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a primosome disassembly mechanism or a new DNA looping mechanism. A fused gp61-gp41 primosome unwinds and primes DNA exclusively via the DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms by disfavoring primosome disassembly, thereby increasing primase processivity. In contrast to T4, priming in bacteriophage T7 and Escherichia coli involves discrete pausing of the primosome and dissociation of the primase from the helicase, respectively. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within primosomes.

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Figure 1: Models of primosome behavior during primer synthesis.
Figure 2: Primer synthesis by primosome depends on rNTP concentration.
Figure 3: Primosome disassembly model for primer synthesis.
Figure 4: DNA looping mechanism for primer synthesis.
Figure 5: Polymerase accessory proteins increase DNA looping.
Figure 6: Model for the primosome activity and initiation of lagging strand DNA synthesis.

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Acknowledgements

We thank T. Lionnet for initial discussions and D. Bensimon for critical reading of the manuscript. This work was supported by a Human Frontier Science Program grant (to V.C. and S.J.B.), a BioNanoSwitch CEE grant (to V.C.) and US National Institutes of Health grant GM013306 (to S.J.B.).

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Author notes

  1. Zhihao Zhuang

    Present address: Present address: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA.,

  2. Maria Manosas and Michelle M Spiering: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratoire de Physique Statistique, Ecole Normale Supérieure, Université Pierre et Marie Curie Paris 06, Université Paris Diderot, Centre National de la Recherche Scientifique, Paris, France

    Maria Manosas & Vincent Croquette

  2. Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA

    Michelle M Spiering, Zhihao Zhuang & Stephen J Benkovic

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  1. Maria Manosas
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  4. Stephen J Benkovic
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Contributions

M.M. contributed to experimental design, performed single-molecule experiments and wrote the manuscript. M.M.S. contributed to experimental design, prepared the DNA substrates and proteins, performed bulk assays and wrote the manuscript. Z.Z. constructed the fusion protein and performed bulk assays. S.J.B. and V.C. wrote the manuscript, and V.C. built the magnetic tweezers.

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Correspondence to Stephen J Benkovic or Vincent Croquette.

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Supplementary Figures 1–9 and Supplementary Methods (PDF 1997 kb)

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Manosas, M., Spiering, M., Zhuang, Z. et al. Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome. Nat Chem Biol 5, 904–912 (2009). https://doi.org/10.1038/nchembio.236

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