Targeting of the Fun30 nucleosome remodeller by the Dpb11 scaffold facilitates cell cycle-regulated DNA end resection

  1. Susanne CS Bantele
  2. Pedro Ferreira
  3. Dalia Gritenaite
  4. Dominik Boos
  5. Boris Pfander  Is a corresponding author
  1. Max Planck Institute of Biochemistry, Germany
  2. University Duisburg-Essen, Germany

Abstract

DNA double strand breaks (DSBs) can be repaired by either recombination-based or direct ligation-based mechanisms. Pathway choice is made at the level of DNA end resection, a nucleolytic processing step, which primes DSBs for repair by recombination. Resection is thus under cell cycle control, but additionally regulated by chromatin and nucleosome remodellers. Here we show that both layers of control converge in the regulation of resection by the evolutionarily conserved Fun30/SMARCAD1 remodeller. Yeast Fun30 and human SMARCAD1 are cell cycle-regulated by interaction with the DSB-localized scaffold proteins Dpb11 and TOPBP1, respectively. In yeast this protein assembly additionally comprises the 9-1-1 damage sensor, is involved in localizing Fun30 to damaged chromatin and thus is required for efficient long-range resection of DSBs. Notably, artificial targeting of Fun30 to DSBs is sufficient to bypass the cell cycle regulation of long-range resection, indicating that chromatin remodelling during resection is underlying DSB repair pathway choice.

Article and author information

Author details

  1. Susanne CS Bantele

    DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Pedro Ferreira

    Centre for Medical Biotechnology, University Duisburg-Essen, Essen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Dalia Gritenaite

    DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Dominik Boos

    Centre for Medical Biotechnology, University Duisburg-Essen, Essen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Boris Pfander

    DNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, Martinsried, Germany
    For correspondence
    bpfander@biochem.mpg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2180-5054

Funding

Deutsche Forschungsgemeinschaft (Project Grant,PF794/3-1)

  • Boris Pfander

Max-Planck-Gesellschaft (Grant)

  • Boris Pfander

Fonds der chemischen Industrie (Fellowship)

  • Susanne CS Bantele

NRW Rueckkehrerprogramm from the stae of North-Rhine-Westphalia (Grant)

  • Dominik Boos

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

Copyright

© 2017, Bantele 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

  • 2,634
    views
  • 760
    downloads
  • 49
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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)

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

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

  1. Susanne CS Bantele
  2. Pedro Ferreira
  3. Dalia Gritenaite
  4. Dominik Boos
  5. Boris Pfander
(2017)
Targeting of the Fun30 nucleosome remodeller by the Dpb11 scaffold facilitates cell cycle-regulated DNA end resection
eLife 6:e21687.
https://doi.org/10.7554/eLife.21687

Share this article

https://doi.org/10.7554/eLife.21687

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Julia Shangguan, Ronald S Rock
    Research Article

    Myosin 10 (Myo10) is a motor protein known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the absolute number of Myo10 molecules during the filopodial lifecycle. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. We combined SDS-PAGE densitometry with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it enriches at opposite cellular ends. Hundreds of Myo10s are in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Live-cell movies reveal a dense cluster of over a hundred Myo10 molecules that initiates filopodial elongation. Hundreds of Myo10 molecules continue to accumulate during filopodial growth, but accumulation ceases when retraction begins. Rates of filopodial elongation, second-phase elongation, and retraction are inversely related to Myo10 quantities. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane compartments. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.

    1. Biochemistry and Chemical Biology
    Megan Larmore, Orhi Esarte Palomero ... Paul G DeCaen
    Research Article

    Ion channels are biological transistors that control ionic flux across cell membranes to regulate electrical transmission and signal transduction. They are found in all biological membranes and their conductive state kinetics are frequently disrupted in human diseases. Organelle ion channels are among the most resistant to functional and pharmacological interrogation. Traditional channel protein reconstitution methods rely upon exogenous expression and/or purification from endogenous cellular sources which are frequently contaminated by resident ionophores. Here, we describe a fully synthetic method to assay functional properties of polycystin channels that natively traffic to primary cilia and endoplasmic reticulum organelles. Using this method, we characterize their oligomeric assembly, membrane integration, orientation, and conductance while comparing these results to their endogenous channel properties. Outcomes define a novel synthetic approach that can be applied broadly to investigate channels resistant to biophysical analysis and pharmacological characterization.