During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.
Bibliographical noteFunding Information:
We thank Joon Lee and William Dunphy from the Dunphy laboratory for comments on the manuscript. We thank Shelly Diamond for expert performance and analysis of GFP by flow cytometry. We thank the Fanconi Anemia Research Fund for the FANCD2 antibodies and 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 crosslink repair
- DNA replication
- Double-strand break repair
- Fanconi anemia