The FAN1–FANCD2 complex prevents chromosomal instability and tumorigenesis independent of ICL repair.
Major finding: The FAN1–FANCD2 complex prevents chromosomal instability and tumorigenesis independent of ICL repair.
Mechanism: FAN1 binds to ubiquitinated FANCD2 and regulates progression at stalled replication forks.
Impact: A FAN1 mutation affecting replication fork processing predisposes patients to pancreatic cancer.
Mutations in Fanconi anemia complementation group D2 (FANCD2) underlie the development of Fanconi anemia by inhibiting the repair of interstrand crosslinks (ICL), which block processes such as DNA transcription and replication. ICL repair requires ubiquitination (Ub) of FANCD2 lysine 561, which is bound by the FANCD1/FANCI-associated nuclease 1 (FAN1). However, the molecular mechanism underlying Ub-FANCD2–mediated ICL repair and the role of FAN1 in the FANCD2 complex remain unknown. Using a mutant FAN1, which cannot bind ubiquitin, and nuclease-defective Fan1nd/nd mouse embryonic fibroblasts (MEF), Lachaud and colleagues showed that FAN1 recruitment by Ub-FANCD2 is not required for ICL repair. However, Fan1nd/nd MEFs exhibited high levels of chromosomal abnormalities in response to replication fork-stalling agents, suggesting that FAN1 nuclease activity prevents chromosomal abnormalities at stalled replication forks without affecting ICL repair. Consistent with these findings, Fancd2-null cells exhibited high levels of chromosomal abnormalities in response to fork-stalling agents, which were not rescued by the non-ubiquitinatable FANCD2 K559R mutant. Double mutant Fan1nd/nd;Fancd2−/− mice had the same level of chromosomal abnormalities in response to fork-stalling agents as single mutants, further supporting the requirement of the FAN1–Ub-FANCD2 complex in preventing chromosomal abnormalities at stalled replication forks. Moreover, DNA fiber analysis indicated that the FAN1–Ub-FANCD2 complex restrains the progression of stalled replication forks. FAN1 mutations predispose to cancer; 85% of Fan1nd/nd mice, but not Fan1+/+ mice, eventually developed tumors, primarily lymphomas and pulmonary carcinomas, suggesting that FAN1 functions as a tumor suppressor via its nuclease activity. Additionally, a recently identified germline FAN1 M50R variant associated with predisposition to high-risk pancreatic cancer and required for FAN1 binding to Ub-FANCD2 resulted in unrestrained fork progression and chromosomal instability. Together, these data show that Ub-FANCD2–mediated recruitment of active FAN1 restrains the progression of stalled replication forks, thereby preventing chromosomal abnormalities, and suggest that FAN1 functions as a tumor suppressor that is essential for genome maintenance.
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