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Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal

Nature volume 391, pages 304–307 (1998)Cite this article

Abstract

DNA polymerases copy DNA templates with remarkably high fidelity, checking for correct base-pair formation both at nucleotide insertion and at subsequent DNA extension steps1,2,3. Despite extensive biochemical, genetic and structural studies2,4 the mechanism by which nucleotides are correctly incorporated is not known. Here we present high-resolution crystal structures of a thermostable bacterial (Bacillus stearothermophilus) DNA polymerase I large fragment5 with DNA primer templates bound productively at the polymerase active site. The active site retains catalytic activity, allowing direct observation of the products of several rounds of nucleotide incorporation. The polymerase also retains its ability to discriminate between correct and incorrectly paired nucleotides in the crystal. Comparison of the structures of successively translocated complexes allows the structural features for the sequence-independent molecular recognition of correctly formed base pairs to be deduced unambiguously. These include extensive interactions with the first four to five base pairs in the minor groove, location of the terminal base pair in a pocket of excellent steric complementarity favouring correct base-pair formation, and a conformational switch from B-form to underwound A-form DNA at the polymerase active site.

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Figure 1: Structure of the Bacillus fragment with duplex DNA bound at the polymerase active site.
Figure 2: Electron-density maps showing catalysis in the crystal.
Figure 3: Polymerase active site.
Figure 4: Polymerase active site with observed DNA and modelled dTTP.

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Acknowledgements

We thank S. Johnson and A. Chapin Rodriguez for assistance in data collection and crystallization, and H. W. Hellinga for discussions. This work was supported by grants to L.S.B. from the American Cancer Society, North Carolina Biotechnology Center, and the Searle Scholars Program.

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Authors and Affiliations

  1. Department of Biochemistry, Duke University Medical Center, Box 3711, Durham, 27710, North Carolina, USA

    James R. Kiefer, Chen Mao & Lorena S. Beese

  2. Stratagene, 11011 N. Torrey Pines Rd, La Jolla, 92017, California, USA

    Jeffrey C. Braman

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  1. James R. Kiefer
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  2. Chen Mao
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  3. Jeffrey C. Braman
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  4. Lorena S. Beese
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Correspondence to Lorena S. Beese.

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Kiefer, J., Mao, C., Braman, J. et al. Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Nature 391, 304–307 (1998). https://doi.org/10.1038/34693

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