Monoubiquitination by the human Fanconi Anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays
- St Vincent's Institute of Medical Research, Australia
- University of Melbourne, Australia
Abstract
FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA using electron microscopy. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
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Funding
National Health and Medical Research Council (GNT1123100 and GNT1181110)
- Andrew J Deans
National Health and Medical Research Council (GNT1156343)
- Wayne M Crismani
Fanconi Anemia Research Fund
- Wayne M Crismani
- Andrew J Deans
Victorian Government's OIS Program
- Winnie Tan
- Sylvie van Twest
- Rohan Bythell-Douglas
- Vincent J Murphy
- Michael Sharp
- Michael W Parker
- Wayne M Crismani
- Andrew J Deans
Maddie Riewoldt's Vision
- Wayne M Crismani
- Andrew J Deans
Victorian Cancer Agency
- Andrew J Deans
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Tan 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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Monoubiquitination by the human Fanconi Anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays
eLife 9:e54128.
https://doi.org/10.7554/eLife.54128
