The Fanconi anemia (FA) gene family comprises at least 12 genes interacting in a 
common pathway involved in DNA repair. To gain insight into the role of FA gene 
inactivation occurring in tumors among the general population, we endogenously 
targeted in cancer cells four FA genes that act at different stages of the FA 
pathway. After successful mono-allelic deletion of all genes, the sequential 
homozygous deletion was achieved only for FANCC and FANCG, acting upstream, but 
not for BRCA2 or FANCD2, acting downstream in the FA pathway. Targeting of the 
second allele in in BRCA2 and FANCD2 heterozygote clones resulted in redeletion 
exclusively of the already defective allele in multiple instances (13x 
concerning BRCA2, 25x concerning FANCD2), strongly suggesting a detrimental 
phenotype. Unlike complete FANCD2 disruption, the mere reduction of FANCD2 
protein levels had no discernible effect. In addition, we confirmed that human 
cancer cells harboring the Seckel ATR mutation display impaired FANCD2 
monoubiquitination and FANCD2 nuclear focus formation, as well as an increased 
sensitivity to DNA interstrand-crosslinking agents. Nevertheless, these cells 
were viable, indicating an ATR-independent function of FANCD2, distinct from its 
major known functions, to be responsible for the detrimental effects of FANCD2 
loss. In conclusion, we established the downstream FA genes FANCD2 and BRCA2 to 
represent particularly vulnerable parts of the FA pathway, providing direct 
evidence for the paradoxical assumption that their inactivation could be 
predominantly selected against in cancer cells. This would explain why certain 
FA gene defects, despite an apparent selection for FA pathway inactivation in 
cancer, are rarely observed in tumors among the general population.