FANCD2 is required for the repair of DNA damage by the FA (Fanconi anemia) pathway, and, consequently, FANCD2-deficient cells are sensitive to compounds such as cisplatin and formaldehyde that induce DNA:DNA and DNA:protein crosslinks, respectively. The DNA2 helicase/nuclease is required for RNA/DNA flap removal from Okazaki fragments during DNA replication and for the resection of DSBs (double-strand breaks) during HDR (homology-directed repair) of replication stress-induced damage. A knockdown of DNA2 renders normal cells as sensitive to cisplatin (in the absence of EXO1) and to formaldehyde (even in the presence of EXO1) as FANCD2-/- cells. Surprisingly, however, the depletion of DNA2 in FANCD2-deficient cells rescues the sensitivity of FANCD2-/- cells to cisplatin and formaldehyde. We previously showed that the resection activity of DNA2 acts downstream of FANCD2 to insure HDR of the DSBs arising when replication forks encounter ICL (interstrand crosslink) damage. The suppression of FANCD2-/- by DNA2 knockdowns suggests that DNA2 and FANCD2 also have antagonistic roles: in the absence of FANCD2, DNA2 somehow corrupts repair. To demonstrate that DNA2 is deleterious to crosslink repair, we used psoralen-induced ICL damage to trigger the repair of a site-specific crosslink in a GFP reporter and observed that "over- resection" can account for reduced repair. Our work demonstrates that excessive resection can lead to genome instability and shows that strict regulatory processes have evolved to inhibit resection nucleases. The suppression of FANCD2-/- phenotypes by DNA2 depletion may have implications for FA therapies and for the use of ICL-inducing agents in chemotherapy.
Bibliographical noteFunding Information:
We thank Dr Bing-hui Shen for access to the DNA2 expression vectors before publication, and Shelley Diamond for expert performance and analysis of GFP by flow cytometry. We thank the Fanconi Anemia Research Fund for the PD20 cell lines. This work was supported by a Breast Cancer grant from Congressionally Directed Medical Research Programs (J.L.C.), GM100186, the Ellison Foundation, and the Ross Fellowship from the Biology Division, California Institute of Technology (K.K.K).
- Dna recombination
- Dna replication
- Fanconi anemia
- Formaldehyde dna damage
- Interstrand crosslinks