RESUMEN
DNA crosslinks block DNA replication and are repaired by the Fanconi anaemia pathway. The FANCD2-FANCI (D2-I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion1. However, D2-I is also known to have a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation2-4. At present, it is 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. Notably, sliding D2-I stalls on encountering single-stranded-double-stranded (ss-ds) DNA junctions, structures that are generated when replication forks stall at DNA lesions5. Using cryogenic electron microscopy, we determined structures of D2-I on DNA that 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 an ssDNA gap, it specifically clamps onto ss-dsDNA junctions. Because 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 the recognition and protection of stalled replication forks in several DNA repair pathways.
Asunto(s)
Daño del ADN , Reparación del ADN , Replicación del ADN , ADN de Cadena Simple , ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Animales , Femenino , Humanos , Extractos Celulares , Microscopía por Crioelectrón , Difusión , ADN/química , ADN/metabolismo , ADN/ultraestructura , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/ultraestructura , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/ultraestructura , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/ultraestructura , Modelos Moleculares , Unión Proteica , Imagen Individual de Molécula , Xenopus laevisRESUMEN
DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2-FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2-FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2-FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2-FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2-FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2-FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair.