1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
Download icon

Single-molecule studies contrast ordered DNA replication with stochastics translesion synthesis

  1. Gengjing Zhao
  2. Emma S Gleave
  3. Meindert Hugo Lamers  Is a corresponding author
  1. Medical Research Council, United Kingdom
Research Article
  • Cited 16
  • Views 2,335
  • Annotations
Cite this article as: eLife 2017;6:e32177 doi: 10.7554/eLife.32177

Abstract

High fidelity replicative DNA polymerases are unable to synthesize past DNA adducts that result from diverse chemicals, reactive oxygen species or UV light. To bypass these replication blocks, cells utilize specialized translesion DNA polymerases that are intrinsically error prone and associated with mutagenesis, drug resistance, and cancer. How untimely access of translesion polymerases to DNA is prevented is poorly understood. Here we use co-localization single-molecule spectroscopy (CoSMoS) to follow the exchange of the E. coli replicative DNA polymerase Pol IIIcore with the translesion polymerases Pol II and Pol IV. We find that in contrast to the toolbelt model, the replicative and translesion polymerases do not form a stable complex on one clamp but alternate their binding. Furthermore, while the loading of clamp and Pol IIIcore is highly organized, the exchange with the translesion polymerases is stochastic and is not determined by lesion-recognition but instead a concentration-dependent competition between the polymerases.

Article and author information

Author details

  1. Gengjing Zhao

    MRC Laboratory of Molecular Biology, Medical Research Council, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Emma S Gleave

    MRC Laboratory of Molecular Biology, Medical Research Council, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Meindert Hugo Lamers

    MRC Laboratory of Molecular Biology, Medical Research Council, Cambridge, United Kingdom
    For correspondence
    m.h.lamers@lumc.nl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4205-1338

Funding

Medical Research Council (U105197143)

  • Gengjing Zhao
  • Emma S Gleave

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Taekjip Ha, Johns Hopkins University School of Medicine, United States

Publication history

  1. Received: September 21, 2017
  2. Accepted: December 5, 2017
  3. Accepted Manuscript published: December 6, 2017 (version 1)
  4. Accepted Manuscript updated: December 7, 2017 (version 2)
  5. Accepted Manuscript updated: December 11, 2017 (version 3)
  6. Version of Record published: December 15, 2017 (version 4)

Copyright

© 2017, Zhao 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

  • 2,335
    Page views
  • 462
    Downloads
  • 16
    Citations

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

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)

  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    Gajanan S Patil et al.
    Research Article Updated

    Fatty acyl-AMP ligases (FAALs) channelize fatty acids towards biosynthesis of virulent lipids in mycobacteria and other pharmaceutically or ecologically important polyketides and lipopeptides in other microbes. They do so by bypassing the ubiquitous coenzyme A-dependent activation and rely on the acyl carrier protein-tethered 4′-phosphopantetheine (holo-ACP). The molecular basis of how FAALs strictly reject chemically identical and abundant acceptors like coenzyme A (CoA) and accept holo-ACP unlike other members of the ANL superfamily remains elusive. We show that FAALs have plugged the promiscuous canonical CoA-binding pockets and utilize highly selective alternative binding sites. These alternative pockets can distinguish adenosine 3′,5′-bisphosphate-containing CoA from holo-ACP and thus FAALs can distinguish between CoA and holo-ACP. These exclusive features helped identify the omnipresence of FAAL-like proteins and their emergence in plants, fungi, and animals with unconventional domain organizations. The universal distribution of FAALs suggests that they are parallelly evolved with FACLs for ensuring a CoA-independent activation and redirection of fatty acids towards lipidic metabolites.

    1. Biochemistry and Chemical Biology
    Urszula Nowicka et al.
    Research Article Updated

    Mitochondria are organelles with their own genomes, but they rely on the import of nuclear-encoded proteins that are translated by cytosolic ribosomes. Therefore, it is important to understand whether failures in the mitochondrial uptake of these nuclear-encoded proteins can cause proteotoxic stress and identify response mechanisms that may counteract it. Here, we report that upon impairments in mitochondrial protein import, high-risk precursor and immature forms of mitochondrial proteins form aberrant deposits in the cytosol. These deposits then cause further cytosolic accumulation and consequently aggregation of other mitochondrial proteins and disease-related proteins, including α-synuclein and amyloid β. This aggregation triggers a cytosolic protein homeostasis imbalance that is accompanied by specific molecular chaperone responses at both the transcriptomic and protein levels. Altogether, our results provide evidence that mitochondrial dysfunction, specifically protein import defects, contributes to impairments in protein homeostasis, thus revealing a possible molecular mechanism by which mitochondria are involved in neurodegenerative diseases.