Final Report Summary - FANDAMAGE (Functional characterization of FAN1, a structure-specific DNA repair nuclease)
DNA inter-strand crosslinks (ICLs) are toxic lesions that covalently link the two DNA strands, thereby preventing their separation during processes such as transcription and DNA replication. Defects in the repair of ICLs in humans causes Fanconi anemia (FA), a rare chromosome instability syndrome characterized by aplastic anemia, developmental abnormalities and high risk of developing cancers. Cells from FA patients are hypersensitive to ICL-inducing agents such as mitomycin C or diepoxybutane, and display a high level of chromosome abnormalities. A major mode of ICL repair relies on the collision of replication fork with the ICL. The products of the FA genes are essential for ICL repair, and a central component in the FA pathway is FANCD2, which is mono–ubiquitinated in S–phase and in response to ICLs. Although we know that this modification is catalysed by the FA core complex that acts as an E3 ubiquitin ligase, how FANCD2 ubiquitination promotes DNA repair remains unclear.
We and others previously described that the FAN1 nuclease is recruited to sites of replication-dependent ICL repair by a mechanism requiring FANCD2 ubiquitination and an UBZ-type ubiquitin binding domain in FAN1. Additionally, depletion or deletion of FAN1 in human cells or in worms causes hypersensitivity to ICL-inducing agents suggesting that the recruitment of FAN1 could be responsible for the ICL repair activity of FANCD2 ubiquitination. From this point of view, FAN1 can be considered as a FA gene. However, biallelic FAN1 mutations do not cause FA in humans but induce karyomegalic interstitial nephritis (KIN), a chronic kidney disease. This observation led me to question the relevance of the FAN1-FANCD2 interaction in the context of ICL repair. Surprisingly, a mutation in the FAN1 UBZ domain, which abolishes FANCD2-dependent recruitment to ICLs in S-phase, does not affect ICL repair. Further experiments led to conclude that FAN1 together with FANCD2 has a new unanticipated role in controlling replication fork stability and chromosome integrity in response to general replication stress. Strikingly, we have identify mutation in FAN1 domain allowing the separation of the replication stress function from the ICL repair function. Moreover, nuclease defective FAN1 knockin mice recapitulate the symptoms of human KIN. Finally, strong evidences indicate a role of replication stress in inducing cancers in Fanconi Anemia, in which context FAN1 behave as a tumour suppressor gene.
Altogether, these results demonstrate that although FAN1 mutation does not lead to anemia, this nuclease acts in a different pathway than the Fanconi Anemia pathway.
We and others previously described that the FAN1 nuclease is recruited to sites of replication-dependent ICL repair by a mechanism requiring FANCD2 ubiquitination and an UBZ-type ubiquitin binding domain in FAN1. Additionally, depletion or deletion of FAN1 in human cells or in worms causes hypersensitivity to ICL-inducing agents suggesting that the recruitment of FAN1 could be responsible for the ICL repair activity of FANCD2 ubiquitination. From this point of view, FAN1 can be considered as a FA gene. However, biallelic FAN1 mutations do not cause FA in humans but induce karyomegalic interstitial nephritis (KIN), a chronic kidney disease. This observation led me to question the relevance of the FAN1-FANCD2 interaction in the context of ICL repair. Surprisingly, a mutation in the FAN1 UBZ domain, which abolishes FANCD2-dependent recruitment to ICLs in S-phase, does not affect ICL repair. Further experiments led to conclude that FAN1 together with FANCD2 has a new unanticipated role in controlling replication fork stability and chromosome integrity in response to general replication stress. Strikingly, we have identify mutation in FAN1 domain allowing the separation of the replication stress function from the ICL repair function. Moreover, nuclease defective FAN1 knockin mice recapitulate the symptoms of human KIN. Finally, strong evidences indicate a role of replication stress in inducing cancers in Fanconi Anemia, in which context FAN1 behave as a tumour suppressor gene.
Altogether, these results demonstrate that although FAN1 mutation does not lead to anemia, this nuclease acts in a different pathway than the Fanconi Anemia pathway.