FANCD2 restrains fork progression and prevents fragility at early origins upon re-replication.

DNA replication is tightly regulated to ensure a single round of chromosome duplication per cell division. DNA licensing restricts origin firing to once-per-cell-cycle while aberrant licensing promotes re-replication and genome instability. Here, we investigate the mechanisms that protect genome integrity following re-replication induced by depletion of the licensing inhibitor Geminin. We find that re-replicating cells require FANCD2 to prevent genome instability. FANCD2 is rapidly recruited to chromatin upon Geminin loss, where it limits unrestrained fork progression and prevents single strand DNA gap accumulation and fork breakage. Genome-wide analyses reveal that upon re-replication, FANCD2 localizes to early origins within highly transcribed regions prone to accumulate R-loops and enriched in early replicating fragile sites. Importantly, reducing transcription and R-loops alleviates re-replication-induced genome fragility, whereas PARP inhibition exacerbates it. Our study uncovers a role for FANCD2 in safeguarding genome integrity during re-replication, offering avenues for selective targeting of cancer cells.