Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within theFRAXAlocus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur atFRAXAfollowing folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS atFRAXAleads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication ofFRAXAunder folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.

Oncogene activation creates DNA replication stress (RS) in cancer cells, which can generate under-replicated DNA regions (UDRs) that persist until cells enter mitosis. UDRs also have the potential to generate DNA bridges in anaphase cells or micronuclei in the daughter cells, which could promote genomic instability. To suppress such damaging changes to the genome, human cells have developed a strategy to conduct ‘unscheduled’ DNA synthesis in mitosis (termed MiDAS) that serves to rescue under-replicated loci. Previous studies have shown that MiDAS proceeds via a POLD3-dependent pathway that shows some features of break-induced replication. Here, we define how human cells utilize both DNA gap filling (REV1 and Pol ζ) and replicative (Pol δ) DNA polymerases to complete genome duplication following a perturbed S-phase. We present evidence for the existence of a polymerase-switch during MiDAS that is required for new DNA synthesis at UDRs. Moreover, we reveal that, upon oncogene activation, cancer cell survival is significantly compromised when REV1 is depleted, suggesting that REV1 inhibition might be a feasible approach for the treatment of some human cancers.

Folate deficiency is associated with a broad range of human disorders, including anemia, fetal neural tube defects, age-associated dementia and several types of cancer. It is well established that a subgroup of rare fragile sites (RFSs) containing expanded CGG trinucleotide repeat (TNR) sequences display instability when cells are deprived of folate. However, given that folate sensitive RFSs exist in a very small percentage of the population, they are unlikely to be the cause of the widespread health problems associated with folate deficiency. We hypothesized that folate deficiency could specifically affect DNA replication at regions containing CG-rich repeat sequences. For this, we identified a region on human chromosome 2 (Chr2) comprising more than 300 CG-rich TNRs (termed “FOLD1”) by examining the human genome database. Via the analysis of chromosome shape and segregation in mitosis, we demonstrate that, when human cells are cultured under folate stress conditions, Chr2 is prone to display a “kink” or “bending” at FOLD1 in metaphase and nondisjunction in anaphase. Furthermore, long-term folate deprivation causes Chr2 aneuploidy. Our results provide new evidence on the abnormalities folate deficiency could cause in human cells. This could facilitate future studies on the deleterious health conditions associated with folate deficiency.