XLF acts as a flexible connector during non-homologous end joining
- Harvard Medical School, United States
- Beckman Research Institute of the City of Hope, United States
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
Felicia Wednesday Lopezcolorado
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.
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.
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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XLF acts as a flexible connector during non-homologous end joining
eLife 9:e61920.
https://doi.org/10.7554/eLife.61920
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