Abstract
The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination. We uncover how the phosphorylation events are tightly regulated in cells and that mimicking their constant phosphorylation leads to an uncontrolled active state of FANCD2, which is loaded onto chromosomes in an unrestrained fashion. Taken together, we describe a mechanism where ATR triggers FANCD2/FANCI loading onto chromosomes.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 112721 |
| Tidsskrift | Cell Reports |
| Vol/bind | 42 |
| Udgave nummer | 7 |
| Antal sider | 21 |
| ISSN | 2211-1247 |
| DOI | |
| Status | Udgivet - 2023 |
| Udgivet eksternt | Ja |
Bibliografisk note
Funding Information:The authors would like to thank members of the Cohn laboratory for reading and discussing the manuscript. We are grateful to staff at the Micron microscopy facility for excellent help and support and to the Advanced Proteomics Facility for mass spectrometry analysis. We thank the Dunn School of Pathology Proteomics Facility for some of the MS experiments. We thank Luke Lavis (Janelia Research Campus) for providing the JF549 Halo ligand dye and Rob Klose for providing the pHTN-HaloTag-3xNLS-CMV-neo plasmid. This work was supported by grants UF100717 and UF150651 from the Royal Society (M.A.C.); grants 153/092 and 0012197 from John Fell Oxford University Press Research Fund (M.A.C.); grants OCRC 0213-MC and CRUKDF 0715-MC from the OCRC/CR-UK (M.A.C.); a grant from the Medical Research Fund (M.A.C.); grant MR/N021002/1 from the Medical Research Council (M.A.C.); a Wellcome Trust Senior Research Fellowship, 210640/Z/18/Z , (M.A.C.); MRC-Clarendon Scholarship (D.L.-M.); a “ Mutua Madrileña ” Graduate Scholarship (D.L.-M.); a Postdoctoral Fellowship from JSPS (Y.Y.); a Wellcome Trust and Royal Society Sir Henry Dale Fellowship 206159/Z/17/Z (S.U.); a Wellcome-Beit Prize 206159/Z/17/B (S.U.); and a Research Prize Fellowship of the Lister Institute of Preventative Medicine (S.U.).
Publisher Copyright:
© 2023 The Author(s)