The Fanconi anemia (FA) pathway preserves genomic stability by coordinating responses to DNA interstrand crosslinks (ICLs) and replication stress. This study examines the role of the ERCC1/XPF endonuclease in FA pathway activation. Using ERCC1-depleted human fibroblasts, we show that ERCC1 is not required for double-strand break formation following exposure to MMC, DEB, or HU, but is essential for FANCD2 monoubiquitination. ERCC1 depletion also reduces radial chromosome formation when combined with FANCA deficiency. These findings place ERCC1 downstream of H2AX phosphorylation and upstream of FA pathway activation, suggesting the FA pathway responds to replication-associated intermediates rather than double-strand breaks.
