FANCD2, FANCJ and BRCA2 cooperate to promote replication fork recovery independently of the Fanconi Anemia core complex

Abstract

Fanconi Anemia (FA) is an inherited multi-gene cancer predisposition syndrome that is characterized on the cellular level by a hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA pathway proteins are thought to act in a linear hierarchy: Following ICL detection, an upstream FA core complex monoubiquitinates the central FA pathway members FANCD2 and FANCI, followed by their recruitment to chromatin. Chromatin-bound monoubiquitinated FANCD2 and FANCI subsequently coordinate DNA repair factors including the downstream FA pathway members FANCJ and FANCD1/BRCA2 to repair the DNA ICL. Importantly, we recently showed that FANCD2 has additional independent roles: it binds chromatin and acts in concert with the BLM helicase complex to promote the restart of aphidicolin (APH)-stalled replication forks, while suppressing the firing of new replication origins. Here, we show that FANCD2 fulfills these roles independently of the FA core complex-mediated monoubiquitination step. Following APH treatment, nonubiquitinated FANCD2 accumulates on chromatin, recruits the BLM complex, and promotes robust replication fork recovery regardless of the absence or presence of a functional FA core complex. In contrast, the downstream FA pathway members FANCJ and BRCA2 share FANCD2's role in replication fork restart and the suppression of new origin firing. Our results support a non-linear FA pathway model at stalled replication forks, where the nonubiquitinated FANCD2 isoform – in concert with FANCJ and BRCA2 – fulfills a specific function in promoting efficient replication fork recovery independently of the FA core complex.

Keywords:

• Fanconi Anemia
• FA pathway
• FANCD2
• FANCD2 monoubiquitination
• FANCJ
• FANCD1/BRCA2
• replication fork recovery

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank the patients with FA and families, the Fanconi Anemia Research Fund, Inc.., and German family organizations “Aktionskreis FA e.V.” and “Deutsche FA Hilfe e.V.” for their support of our research. We are grateful to F. Rosselli, G. Kupfer and J. Chen for sharing human cell lines with us. We are grateful to A. Auerbach (New York, NY) and D. Schindler (Würzburg, Germany) for providing FA fibroblast cells and for sharing the mutation data that confirmed our retroviral complementation results. We would like to thank N. Shima for careful reading of the manuscript and helpful discussions.

Funding

AS was supported by the National Science Foundation (award 1121023) and the American Cancer Society (RSG-13-039-01-DMC). HH was supported by the NIH R01 CA155294-04 and by the BMBF grant “FoneFA.” In addition, this work was also partly supported by a Brainstorm Award from the Masonic Cancer Center at the University of Minnesota.

Author Contributions

MR co-planned and performed the experiments and co-wrote the manuscript. IC performed some of the DNA fiber assays. SLK and HH generated human immortalized FA patient cell lines and complemented FA patient cell lines. AS co-planned the experiments and co-wrote the manuscript.

Supplemental Material

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