Markus Räschle

Fanconi anemia is a human cancer predisposition syndrome caused by mutations in 13 Fanc genes. The disorder is characterized by genomic instability and cellular hypersensitivity to chemicals that generate DNA interstrand cross-links (ICLs). A central event in the activation of the Fanconi anemia pathway is the mono-ubiquitylation of the FANCI-FANCD2 complex, but how this complex confers ICL resistance remains enigmatic. Using a cell-free system, we showed that FANCI-FANCD2 is required for replication-coupled ICL repair in S phase. Removal of FANCD2 from extracts inhibits both nucleolytic incisions near the ICL and translesion DNA synthesis past the lesion. Reversal of these defects requires ubiquitylated FANCI-FANCD2. Our results show that multiple steps of the essential S-phase ICL repair mechanism fail when the Fanconi anemia pathway is compromised.

DNA interstrand cross-links (ICLs) are toxic DNA lesions whose repair in S phase of eukaryotic cells is incompletely understood. In Xenopus egg extracts, ICL repair is initiated when two replication forks converge on the lesion. Dual incisions then create a DNA double-strand break (DSB) in one sister chromatid, whereas lesion bypass restores the other sister. We report that the broken sister chromatid is repaired via RAD51-dependent strand invasion into the regenerated sister. Recombination acts downstream of FANCI-FANCD2, yet RAD51 binds ICL-stalled replication forks independently of FANCI-FANCD2 and before DSB formation. Our results elucidate the functional link between the Fanconi anemia pathway and the recombination machinery during ICL repair. In addition, they demonstrate the complete repair of a DSB via homologous recombination in vitro.