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Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale

Nature Structural & Molecular Biology volume 23, pages 402408 (2016) | Download Citation

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Abstract

DNA polymerase delta (Pol δ) is responsible for elongation and maturation of Okazaki fragments. Pol δ and the flap endonuclease FEN1, coordinated by the PCNA clamp, remove RNA primers and produce ligatable nicks. We studied this process in the Saccharomyces cerevisiae machinery at millisecond resolution. During elongation, PCNA increased the Pol δ catalytic rate by >30-fold. When Pol δ invaded double-stranded RNA–DNA representing unmatured Okazaki fragments, the incorporation rate of each nucleotide decreased successively to 10–20% that of the preceding nucleotide. Thus, the nascent flap acts as a progressive molecular brake on the polymerase, and consequently FEN1 cuts predominantly single-nucleotide flaps. Kinetic and enzyme-trapping experiments support a model in which a stable PCNA–DNA–Pol δ–FEN1 complex moves processively through iterative steps of nick translation, ultimately completely removing primer RNA. Finally, whereas elongation rates are under dynamic dNTP control, maturation rates are buffered against changes in dNTP concentrations.

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Change history

  • 19 April 2016

    In the version of this article initially published online, there were partial omissions within the schematics depicted in Figure 5a,b. These errors have been corrected for the print, HTML and PDF versions of the article.

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Acknowledgements

The authors thank J. Majors, R. Galletto, and T. Lohman for critical discussions during the progress of this work, and C. Stith for protein purification. This work was supported in part by the US National Institutes of Health (GM032431 to P.B.) and from the US-Israel Binational Science Foundation (2013358 to P.B.).

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Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri, USA.

    • Joseph L Stodola
    •  & Peter M Burgers

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Contributions

J.L.S. and P.M.B. designed experiments and analyzed data. J.L.S. performed all experiments. J.L.S. and P.M.B. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Peter M Burgers.

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https://doi.org/10.1038/nsmb.3207