Abstract

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we use Xenopus laevis egg extract to investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

Data availability

Source data files for all summary graphs have been provided. The MATLAB scripts used to analyze and generate the results shown in Figure 3 and Supplementary Figure 5 are also included.

Article and author information

Author details

  1. Sean M Carney

    Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2674-1064
  2. Andrew T Moreno

    Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Sadie C Piatt

    Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Metztli Cisneros-Aguirre

    Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, Duarte, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Felicia Wednesday Lopezcolorado

    Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, Duarte, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2916-6042
  6. Jeremy M Stark

    Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, Duarte, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Joseph J Loparo

    Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    For correspondence
    joseph_loparo@hms.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4941-4696

Funding

National Institutes of Health (R01GM115487)

  • Joseph J Loparo

National Institutes of Health (R01CA197506)

  • Jeremy M Stark

National Institutes of Health (R01CA240392)

  • Jeremy M Stark

National Institutes of Health (F32GM129913)

  • Sean M Carney

National Institutes of Health (T32 GM008313)

  • Sadie C Piatt

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

Reviewing Editor

  1. Maria Spies, University of Iowa, United States

Ethics

Animal experimentation: The Institutional Animal Care and Use Committee (IACUC) of Harvard Medical School approved of all work performed in this study (Protocol# IS00000051-6), which was done in accordance with AAALAC rules and regulations.

Version history

  1. Received: August 8, 2020
  2. Accepted: December 7, 2020
  3. Accepted Manuscript published: December 8, 2020 (version 1)
  4. Version of Record published: December 16, 2020 (version 2)

Copyright

© 2020, Carney 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.

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  1. Sean M Carney
  2. Andrew T Moreno
  3. Sadie C Piatt
  4. Metztli Cisneros-Aguirre
  5. Felicia Wednesday Lopezcolorado
  6. Jeremy M Stark
  7. Joseph J Loparo
(2020)
XLF acts as a flexible connector during non-homologous end joining
eLife 9:e61920.
https://doi.org/10.7554/eLife.61920

Share this article

https://doi.org/10.7554/eLife.61920

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