1. Structural Biology and Molecular Biophysics
Download icon

cryo-EM structures of the E. coli replicative DNA polymerase reveal dynamic interactions with clamp, exonuclease and τ

  1. Rafael Fernández-Leiro
  2. Julian Conrad
  3. Sjors HW Scheres
  4. Meindert Hugo Lamers  Is a corresponding author
  1. MRC laboratory of Molecular Biology, United Kingdom
  2. Medical Research Council Laboratory of Molecular Biology, United Kingdom
Research Article
  • Cited 39
  • Views 6,097
  • Annotations
Cite this article as: eLife 2015;4:e11134 doi: 10.7554/eLife.11134

Abstract

The replicative DNA polymerase PolIIIα from E. coli is a uniquely fast and processive enzyme. For its activity it relies on the DNA sliding clamp β, the proofreading exonuclease ε and the C-terminal domain of the clamp loader subunit τ. Due to the dynamic nature of the four-protein complex it has long been refractory to structural characterization. Here we present the 8 Å resolution cryo-electron microscopy structures of DNA-bound and DNA-free states of the PolIII-clamp-exonuclease-τc complex. The structures show how the polymerase is tethered to the DNA through multiple contacts with the clamp and exonuclease. A novel contact between the polymerase and clamp is made in the DNA bound state, facilitated by a large movement of the polymerase tail domain and τc. These structures provide crucial insights into the organization of the catalytic core of the replisome and form an important step towards determining the structure of the complete holoenzyme.

Article and author information

Author details

  1. Rafael Fernández-Leiro

    Structural Studies, MRC laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  2. Julian Conrad

    Structural Studies, MRC laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  3. Sjors HW Scheres

    Structural Studies, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    Sjors HW Scheres, Reviewing editor, eLife.
  4. Meindert Hugo Lamers

    Structural Studies, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom
    For correspondence
    mlamers@mrc-lmb.cam.ac.uk
    Competing interests
    No competing interests declared.

Reviewing Editor

  1. Stephen C Kowalczykowski, University of California, Davis, United States

Publication history

  1. Received: August 25, 2015
  2. Accepted: October 23, 2015
  3. Accepted Manuscript published: October 24, 2015 (version 1)
  4. Version of Record published: December 9, 2015 (version 2)

Copyright

© 2015, Fernández-Leiro et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 6,097
    Page views
  • 2,325
    Downloads
  • 39
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Computational and Systems Biology
    2. Structural Biology and Molecular Biophysics
    Alexandra Kühnlein et al.
    Research Article

    Can replication and translation emerge in a single mechanism via self-assembly? The key molecule, transfer RNA (tRNA), is one of the most ancient molecules and contains the genetic code. Our experiments show how a pool of oligonucleotides, adapted with minor mutations from tRNA, spontaneously formed molecular assemblies and replicated information autonomously using only reversible hybridization under thermal oscillations. The pool of cross-complementary hairpins self-selected by agglomeration and sedimentation. The metastable DNA hairpins bound to a template and then interconnected by hybridization. Thermal oscillations separated replicates from their templates and drove an exponential, cross-catalytic replication. The molecular assembly could encode and replicate binary sequences with a replication fidelity corresponding to 85–90 % per nucleotide. The replication by a self-assembly of tRNA-like sequences suggests that early forms of tRNA could have been involved in molecular replication. This would link the evolution of translation to a mechanism of molecular replication.

    1. Chromosomes and Gene Expression
    2. Structural Biology and Molecular Biophysics
    Samuel Bowerman et al.
    Research Article

    Eukaryotes and many archaea package their DNA with histones. While the four eukaryotic histones wrap ~147 DNA base pairs into nucleosomes, archaeal histones form 'nucleosome-like' complexes that continuously wind between 60 - 500 base pairs of DNA ('archaeasomes'), suggested by crystal contacts and analysis of cellular chromatin. Solution structures of large archaeasomes (>90 DNA base pairs) have never been directly observed. Here, we utilize molecular dynamics simulations, analytical ultracentrifugation, and cryoEM to structurally characterize the solution state of archaeasomes on longer DNA. Simulations reveal dynamics of increased accessibility without disruption of DNA-binding or tetramerization interfaces. Mg2+ concentration influences compaction, and cryoEM densities illustrate that DNA is wrapped in consecutive substates arranged 90o out-of-plane with one another. Without ATP-dependent remodelers, archaea may leverage these inherent dynamics to balance chromatin packing and accessibility.