Monoubiquitination by the human Fanconi Anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays
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.
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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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