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Cryo-EM structure and dynamics of eukaryotic DNA polymerase δ holoenzyme

Nature Structural & Molecular Biology volume 26, pages 955–962 (2019)Cite this article

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Abstract

DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. Saccharomyces cerevisiae Polδ consists of catalytic Pol3 and the regulatory Pol31 and Pol32 subunits. Here, we present the near atomic resolution (3.2 Å) cryo-EM structure of yeast Polδ holoenzyme in the act of DNA synthesis. The structure reveals an unexpected arrangement in which the regulatory subunits (Pol31 and Pol32) lie next to the exonuclease domain of Pol3 but do not engage the DNA. The Pol3 C-terminal domain contains a 4Fe−4S cluster and emerges as the keystone of Polδ assembly. We also show that the catalytic and regulatory subunits rotate relative to each other and that this is an intrinsic feature of the Polδ architecture. Collectively, the structure provides a framework for understanding DNA transactions at the replication fork.

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Fig. 1: Cryo-EM structure of DNA bound Polδ holoenzyme.
Fig. 2: Structure and cryo-EM density for selected regions of catalytic and regulatory modules and CysBD.
Fig. 3: CysBD is the keystone of Polδ assembly.
Fig. 4: Flexibility between catalytic and regulatory modules.

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Data availability

The cryo-EM density map has been deposited in the Electron Microscopy Data Bank under accession code EMD-20235. Atomic coordinates have been deposited in the Protein Data Bank (https://www.rcsb.org) with accession code 6P1H. All reagents and relevant data are available from the authors upon reasonable request.

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Acknowledgements

We thank B. Carragher, C. Potter and E. Eng for helpful advice and discussions throughout the project. Some of the work was supported by grant GM129689 from the NIH (S.P.). Initial EM screening was performed at the Icahn School of Medicine microscope facility supported by a shared instrumentation grant from the NIH (1S10RR026473). Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR and the NIH National Institute of General Medical Sciences (GM103310), with additional support from Agouron Institute (F00316), NIH (OD019994) and NIH (RR029300). Computing resources needed for this work were provided in part by the High Performance Computing facility of the Icahn School of Medicine at Mount Sinai. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.

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Authors and Affiliations

  1. Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Rinku Jain, Radhika Malik, Iban Ubarretxena-Belandia & Aneel K. Aggarwal

  2. Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA

    William J. Rice

  3. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA

    Robert E. Johnson, Louise Prakash & Satya Prakash

  4. Instituto Biofisika (UPV/EHU, CSIC), University of the Basque Country, Leioa, Spain

    Iban Ubarretxena-Belandia

  5. Ikerbasque, Basque Foundation for Science, Bilbao, Spain

    Iban Ubarretxena-Belandia

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  1. Rinku Jain
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Contributions

A.K.A. and R.J. conceived the project and designed the experiments. R.E.J. expressed the complex in yeast. R.J. purified the complex for cryo-EM. R.M. prepared the grids. R.J. and W.J.R. collected the data and reconstructed the 3D structures. R.J. built and refined the atomic models. A.K.A. guided the overall project, I.U.-B. guided some of the cryo-EM experiments, and S.P. and L.P. guided the protein expression studies. R.J. and A.K.A. prepared the manuscript, with input from all the authors.

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Correspondence to Rinku Jain or Aneel K. Aggarwal.

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The authors declare no competing interests.

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Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Cryo-EM image processing.

Particles from three different data sets were merged and 3D refinement was performed with both cryoSPARC and RELION 3.0 beta to yield consensus maps at nominal resolution of 3.2 and 3.4 Å respectively. Consensus map from RELION was subjected to multi-body refinement, resulting in major improvement in density for Pol32N (circled in red) and regions of Pol31 that are furthest from the catalytic module.

Supplementary Figure 2 Comparison of Polδ regulatory module and CysBD with those in homologs.

a, The CysBD interacting interface of the regulatory module is substantially different between yeast Polδ, human Polδ, human Polα and human Polε. b, CysBD of yeast Polδ is much smaller in size than the counterparts from pols α and ε. CysAD is disordered in yeast Polδ.

Supplementary Figure 3 Model of Polδ–PCNA complex.

Model of yeast Polδ (this work) and PCNA (PDB 2OD8) derived by threading the Polδ bound DNA through the central hole of the PCNA.

Supplementary Figure 4 CysBD mediated changes in the Pol3 catalytic module.

a, CysBD draws the exo and thumb domains of the catalytic module closer than their positions in the structure of the isolated catalytic module (3IAY) and results in an overall compaction of the catalytic module by >2.5 Å. b, A primer modeled at the exo active side is within van der Waals distance from Tyr496, which is part of the exo domain loop encompassing amino acids 490–497 that interacts with CysBD. This loop is disordered in the structure of the isolated catalytic module (3IAY).

Supplementary Figure 5 Multi-body refinement and principal component analysis of the relative orientations of the catalytic and regulatory modules.

a, Consensus and multi-body maps colored by local resolution. Multi-body analysis results in a substantial improvement in resolution for regions of Pol31 and Pol32 that are farthest from the catalytic module, for example, Pol32N and the OB fold of Pol31. b, Contribution of all eigenvectors to the variance. c, Histograms of amplitudes along the first two eigenvectors. Both histograms are unimodal, indicating continuous motion.

Supplementary Figure 6 Trajectories from normal mode analysis compared to motion from multi-body analysis.

End of range conformations for the regulatory module are shown in green and red. The first eigenvector from principal component analysis of multi-body refinement represents rocking motion of the regulatory module parallel to the catalytic module (middle), while the second eigenvector represents rocking motion towards the catalytic module (bottom). Trajectories for the first non-trivial mode of molecular motion from normal mode analysis (top) correspond to the motion represented by the first eigenvector (middle).

Supplementary Figure 7 Disease mutations in human Polδ.

Cancer driver mutations in human Polδ mapped on the structure of yeast Polδ (top). These mutations are distributed on the NTD, exo, palm, fingers and CysAD domains. The oncogenic R506H (exo) mutation and the MDPL associated R507C (exo) and I1070N (CysBD) mutations map to the interface between the catalytic and regulatory modules.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7.

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Supplementary Video 1

Motion represented by the first eigenvector from multi-body refinement. Motion represented by the first eigenvector corresponds to a rocking motion of the regulatory module parallel to the catalytic module. The DNA bound catalytic module is shown in cyan and CysBD and the regulatory module are in green.

Supplementary Video 2

Motion represented by the first eigenvector from multi-body refinement. Motion represented by the first eigenvector corresponds to a rocking motion of the regulatory module parallel to the catalytic module. DNA bound catalytic module is shown in cyan and CysBD and the regulatory module are in green.

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Jain, R., Rice, W.J., Malik, R. et al. Cryo-EM structure and dynamics of eukaryotic DNA polymerase δ holoenzyme. Nat Struct Mol Biol 26, 955–962 (2019). https://doi.org/10.1038/s41594-019-0305-z

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