Proximal and Distal Interactions of Fanconi Anemia (fa) Protein Fancd2 with Genomic Interstrand Crosslinks (Icls)

Objective Identification of DNA Damage Response (DDR) partners for ICL proximal and distal recruitment of FANCD2 Methods: Laser/confocal microscopy to localize antigen tagged psoralen ICLs in the genome of living cells; the proximity ligation assay (PLA) to identify interactions between FANCD2, DDR proteins, and ICLs. DNA adducts can trigger the DDR, in which many proteins are recruited to genomic chromatin in the vicinity of the damage and the same protein may have multiple partners and functions. The proteins include those engaged in repair, which are proximal to the lesion, while those involved in other pathways may be some distance from the damage. Currently it is not possible to distinguish lesion proximal from distal proteins. FA cells are hypersensitive to agents that form ICLs, and FA proteins are involved in the DDR following replication stress. The monoubiquitinated FANCD2 plays an important role in ICL repair during replication. We synthesized psoralens linked to the immunotag digoxigenin (Dig), and detected, by immunostaining of the tag, laser localized psoralen ICLs. We monitored the recruitment of DDR proteins to the ICLs and unexpectedly found that FANCD2 was a component of the DDR, and was important for ICL repair, in all phases of the cell cycle. By co-IP and PLA we found novel interactions between FANCD2 and DDR proteins. We identified those protein partners important for ICL repair, used PLA to denote the partners required for recruitment of ICL proximal and distal FANCD2, and thus identified the fraction of FANCD2 involved in ICL repair