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Structural basis for processive DNA synthesis by yeast DNA polymerase ɛ

Nature Structural & Molecular Biology volume 21, pages 49–55 (2014)Cite this article

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Abstract

DNA polymerase ɛ (Pol ɛ) is a high-fidelity polymerase that has been shown to participate in leading-strand synthesis during DNA replication in eukaryotic cells. We present here a ternary structure of the catalytic core of Pol ɛ (142 kDa) from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide. This structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ɛ has the highest fidelity among B-family polymerases despite the absence of an extended β-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain that allows Pol ɛ to encircle the nascent double-stranded DNA. Altogether, the structure provides an explanation for the high processivity and high fidelity of leading-strand DNA synthesis in eukaryotes.

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Figure 1: Structure of the Pol ɛ–DNA–dATP ternary complex.
Figure 2: Comparison of the overall structure of Pol ɛ and Pol δ.
Figure 3: Comparison of β-hairpin loops in Pol ɛ, Pol δ and RB69 gp43.
Figure 4: Impact of the P domain on the polymerase activity and processivity of Pol ɛ.
Figure 5: Interactions between waters, bases, and amino acids in the nascent base pair–binding pocket.
Figure 6: Comparison of the protein residues that come to within 10 Å of the DNA in selected DNA polymerases.

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Acknowledgements

Data were collected at beamline ID23-2 of the European Synchrotron Radiation Facility. We thank U.H. Sauer (Umeå University) for help and advice with crystal handling and diffraction-data collection, S. Huang for help with refinement, P. Burgers (Washington University School of Medicine) for the kind gift of purified DNA polymerase δ, the Protein Production Platform of Umeå University for help with protein production and the High Performance Computing Center North (HPC2N) in the Swedish National Strategic e-Science Research Program eSSENCE for their technical support. This research was supported by the Swedish Research Council (E.J. and A.E.S.-E.), Swedish Cancer Society (E.J.), Kempe Foundations (E.J. and A.E.S.-E.), Knut and Alice Wallenberg Foundation (E.J.), Insamlingstiftelsen vid den medicinska fakulteten vid Umeå Universitet (E.J.), Karriärbidrag, Umeå University (E.J.) and Umeå Centre for Microbial Research (A.E.S.-E.).

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Authors and Affiliations

  1. Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden

    Matthew Hogg, Pia Osterman, Göran O Bylund, Rais A Ganai, Else-Britt Lundström & Erik Johansson

  2. Department of Chemistry, Umeå University, Umeå, Sweden

    A Elisabeth Sauer-Eriksson

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Contributions

E.-B.L. overexpressed and purified the protein. M.H. and P.O. grew and handled the crystals. M.H., A.E.S.-E. and E.J. collected the diffraction data. A.E.S.-E. determined and refined the structure. P.O., G.O.B. and R.A.G. constructed the mutant proteins and performed the enzymatic assays. M.H., A.E.S.-E. and E.J. designed the experiments and prepared the manuscript. All authors discussed and commented on the manuscript.

Corresponding authors

Correspondence to A Elisabeth Sauer-Eriksson or Erik Johansson.

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Integrated supplementary information

Supplementary Figure 1 Localization of insertions in the structure of Pol ɛ.

(a) The ribbon structure of Pol ɛ is colored in green with the insertions that are not present in Pol δ highlighted in gold. (b) To aid the reader, the same orientation of the Pol ɛ structure in (a) is shown but now colored according to its respective domains (see Figure 1 for color coding). (c) A close-up view of the N-terminal domains from four DNA polymerases (Pol ɛ, Pol δ (PDB entry 3iay), Herpes Simplex Virus-I (PDB entry 2gv9) and RB69 gp43 (PDB entry 1ig9)).

Supplementary Figure 2 Schematic diagram showing the interactions between Pol ɛ and the 11–16 primer template DNA.

Residues interacting with DNA are color coded according to the domains they belong to (see Figure 1 for color coding). Residues in brackets indicate that these residues have side chains positioned in the vicinity of the DNA with the potential to form direct salt-bridges to the phosphodiester backbone. The first iodo-cytosine in the template DNA strand is not modeled in the structure and is shaded in white. Three base-specific contacts are indicated: the Nζ atom of K967 makes one base-specific hydrogen bond with the O2 atom of iodo-uracil 10, and the Nη1 and Nη2 atoms of R988 make base-specific hydrogen bonds to the N3 atom of guanine 8 and the O2 atom of iodo-uracil 7, respectively. The three nucleotides mentioned are all located in the primer strand. Water-mediated interactions between amino acids and the template bases are indicated with a red line and shown in Figure 5. The main-chain amino group of Y645 forms a hydrogen bond to the 3'-hydroxyl group on the deoxyribose moiety of the dATP

Supplementary Figure 3 Composition of the polymerase active sites in Pol ɛ, Pol δ (PDB 3IAY) and RB69 gp43 (PDB 1IG9).

Metals and carbon atoms are shown in green, blue, and yellow in Pol ɛ, Pol δ, and RB69 gp43, respectively. Water molecules are shown as red spheres. A superimposition of the three active sites is also shown. The corresponding residues D877 in Pol ɛ, D764 in Pol δ, and D623 in RB69 gp43 are in almost identical positions in all three structures, but the orientations of the corresponding residues D640 in Pol ɛ, D411 in RB69 gp43, and D608 in Pol δ vary.

Supplementary Figure 4 Composition of the P domain and metal-binding sites

(a) Close-up view of the P-domain (dark blue), primer template dsDNA (orange) and dATP (red) in two orientations. (b) Close-up view of Mg2+ ion coordination in Pol ɛ. The Mg2+ ion, bound in site B, is octahedrally coordinated by D640, D877, the main chain carbonyl oxygen of V641, and three oxygen atoms from the triphosphate moiety of dATP. For clarity, the carbon atoms of ddC are shown in orange whereas other carbon atoms are shown in yellow. Some distances in Å, including metal- and hydrogen bonds, and the distance between the 3'-C of the primer-end and the α-phosphate, are shown as dashed lines. (c) The anomalous map showed electron density over the metal site of ~6 sigma over the background noise. The density is contoured at 4 sigma level in the figure.

Supplementary Figure 5 Distribution of cancer-associated mutations in Pol ɛ.

(a-d) Cancer-associated mutations in human POLE have previously been reported. We have indicated the corresponding amino acids in red in Pol ɛ.

Supplementary Figure 6 Antimutator mutations in Pol ɛ previously identified in a genetic screen.

The five amino acid changes isolated in that study are listed in the table and the targeted amino acids are shown in blue in the structure.

Supplementary Figure 7 Original images of gels and autoradiographs presented in Figure 4.

(a) Primer-extension assay presented in Figure 4a. (b) DNA polymerase assay on singly primed, single-stranded Bluescript SKII template presented in Figure 4d.

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Supplementary Figures 1–7 and Supplementary Tables 1–3 (PDF 8027 kb)

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Hogg, M., Osterman, P., Bylund, G. et al. Structural basis for processive DNA synthesis by yeast DNA polymerase ɛ. Nat Struct Mol Biol 21, 49–55 (2014). https://doi.org/10.1038/nsmb.2712

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