Mitotic DNA Synthesis in Untransformed Human Cells Preserves Common Fragile Site Stability via a FANCD2-Driven Mechanism That Requires HELQ

[Display omitted] •Untransformed human cells operate MiDAS that depends on FANCD2 but not RAD52.•This form of MiDAS requires FANCD2 mono-ubiquitination as a prerequisite step.•HELQ and FANCD2 (but not RAD52) act epistatically in supporting MiDAS.•Unlike cancer cells, untransformed cells use MiDAS to protect common fragile sites.•MiDAS in untransformed cells is functionally distinct from that in cancer cells. Faithful genome duplication is a challenging task for dividing mammalian cells, particularly under replication stress where timely resolution of late replication intermediates (LRIs) becomes crucial prior to cell division. In human cancer cells, mitotic DNA repair synthesis (MiDAS) is described as a final mechanism for the resolution of LRIs to avoid lethal chromosome mis-segregation. RAD52-driven MiDAS achieves this mission in part by generating gaps/breaks on metaphase chromosomes, which preferentially occur at common fragile sites (CFS). We previously demonstrated that a MiDAS mechanism also exists in untransformed and primary human cells, which is RAD52 independent but requires FANCD2. However, the properties of this form of MiDAS are not well understood. Here, we report that FANCD2-driven MiDAS in untransformed human cells: 1) requires a prerequisite step of FANCD2 mono-ubiquitination by a subset of Fanconi anemia (FA) proteins, 2) primarily acts to preserve CFS stability but not to prevent chromosome mis-segregation, and 3) depends on HELQ, which potentially functions at an early step. Hence, FANCD2-driven MiDAS in untransformed cells is built to protect CFS stability, whereas RAD52-driven MiDAS in cancer cells is likely adapted to prevent chromosome mis-segregation at the cost of CFS expression. Notably, we also identified a novel form of MiDAS, which surfaces to function when FANCD2 is absent in untransformed cells. Our findings substantiate the complex nature of MiDAS and a link between its deficiencies and the pathogenesis of FA, a human genetic disease.