Dissecting cellular inter‐strand crosslink repair pathways through the recruitment kinetics of Fanconi Anemia (FA) proteins to localized ICLs

Interstrand cross‐links (ICLs) are absolute blocks to transcription and replication and can provoke genomic instability and cell death. FA proteins are known to be recruited to ICL blocked replication forks, while their contribution to DNA repair in other phases of the cell cycle remains obscure. We have developed a novel experimental strategy whereby ICLs are introduced into defined subnuclear regions by laser localized photoactivation of psoralen, after which the appearance of repair proteins can be monitored by confocal immunofluorescence microscopy. This technique permits the visualization/quantification of protein recruitment immediately after ICL formation, which is not possible with other approaches. The FA core complex proteins FANCM and FANCE/FANCF are recruited to sites of damage exclusively in S phase in 3 and 5 minutes respectively. FANCD2 localized within 10 to 15 min during all phases of the cell cycle, in a FA core complex dependent fashion. Thus, core complex function, but not necessarily colocalization, was required for FANCD2 recruitment. The FA‐associated Nuclease (FAN1) has been reported to require its Ubiquitin Binding Domain (UBZ) to colocalize with FANCD2. We found two recruitment patterns of FAN1; immediate localization (within 10sec) to ICL sites which was independent of UBZ, followed by a second phase (10 min), which was dependent on the UBZ domain.