The genetic syndrome Fanconi anemia (FA) is characterized by aplastic anemia, 
cancer predisposition and hypersensitivity to DNA interstrand crosslinks (ICLs). 
FA proteins (FANCs) are thought to work in pathway(s) essential for dealing with 
crosslinked DNA. FANCs interact with other proteins involved in both DNA repair 
and S-phase checkpoint such as BRCA1, ATM and the RAD50/MRE11/NBS1 (RMN) 
complex. We deciphered the previously undefined pathway(s) leading to the 
ICLs-induced S-phase checkpoint and the role of FANCs in this process. We found 
that ICLs activate a branched pathway downstream of the ATR kinase: one branch 
depending on CHK1 activity and the other on the FANCs-RMN complex. The transient 
slow-down of DNA synthesis was abolished in cells lacking ATR, whereas 
CHK1-siRNA-treated cells, NBS1 or FA cells showed partial S-phase arrest. CHK1 
RNAi in NBS1 or FA cells abolished the S-phase checkpoint, suggesting that CHK1 
and FANCs/NBS1 proteins work on parallel pathways. Furthermore, we found that 
ICLs trigger ATR-dependent FANCD2 phosphorylation and FANCD2/ATR colocalization. 
This study demonstrates a novel relationship between the FA pathway(s) and the 
ATR kinase.