Alcon-et-al-2024-FANCD2I

DNA crosslinks block DNA replication and are repaired by the Fanconi Anemia pathway. The FANCD2-FANCI (D2-I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion 1. However, D2-I is also known to play a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation 2-4. It is currently unclear how DNA crosslinks are recognized and how D2-I functions in replication fork protection. Here, using single-molecule imaging, we show that D2-I is a sliding clamp that binds to and diffuses on double-stranded DNA. Strikingly, sliding D2-I stalls upon encountering single-stranded–double-stranded (ss-ds) DNA junctions, structures that are generated when replication forks stall at DNA lesions 5. Using cryoEM, we determined structures of D2-I on DNA which show that stalled D2-I makes specific interactions with the ss-dsDNA junction that are distinct from those made by sliding D2-I. Thus, D2-I surveys dsDNA and, when it reaches a ssDNA gap, it specifically clamps onto ss-dsDNA junctions. Since ss-dsDNA junctions are found at stalled replication forks, D2-I can identify sites of DNA damage. Therefore, our data provide a unified molecular mechanism that reconciles the roles of D2-I in recognition and protection of stalled replication forks in multiple DNA repair pathways.