Activation mechanism of the Fanconi anemia pathway following DNA damage

Abstract

Stalling of the replication forks activates ATRIP-ATR kinase complex through the direct association with the activation domain of TopBP1, leading to Chk1 phosphorylation and S-phase checkpoint activation. This has been shown to require the serial actions of Rad17 clamp loader and Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp. Fanconi anemia proteins FANCI and FANCD2 interact with each other, forming nuclear D2-I complex. We have previously reported that multiple phosphorylation events on FANCI protein serve as a molecular switch to activate FANCD2 monoubiquitination mediated by the FA core E3 ligase complex, a critical event in DNA crosslink repair. Here we show that ATRIP is critical for the FA pathway activation. However, contrary to the expectation, Rad17 and TopBP1 were largely dispensable for FANCI phosphorylation as well as FANCD2 monoubiquitination. On the other hand, the core complex components as well as FANCD2 were required for FANCI phosphorylation, indicating their role linking ATR and FANCI. These results indicate the distinct molecular requirements for FANCI phosphorylation compared to other ATR substrates, and a novel mode of ATR activation in the DNA damage response. We also found that ATM was not essential for FANCD2 monoubiquitination in response to IR. However, Ubc13 was required for FANCD2 monoubiquitination/ focus formation similarly to the case of Rad51, suggesting that the end resection is the prerequisite for FA pathway activation mediated by ATR following IR.