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 the FRAXA locus 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 at FRAXA following 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 at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.

叶酸剥夺导致以CGG三核苷酸重复（TNR）序列为特征的一组稀有脆弱位点（RFS）不稳定。FRAXA中TNR的病理扩展基因座干扰DNA复制，是脆性X综合征（与认知障碍相关的性相关疾病）的主要病因。尽管对叶酸敏感的RFS具有共同的易碎位点（CFS；在所有个体中均存在）的许多特征，但它们是由不同的压力诱导的，并且没有序列相似性。已知在早期有丝分裂中采用了一种途径（称为MiDAS）来完成CFS的复制。此过程需要RAD52，并且与癌症中CFS处的移位和拷贝数变化有关。但是，目前还不清楚RFS是否也使用MiDAS，以及共享或不同机制在多大程度上导致CFS和RFS的脆弱性。在这里，我们证明MiDAS确实发生在FRAXA继叶酸剥夺后，通过某种途径进行，该途径表现出与CFS处的机制不同，这取决于RAD51，SLX1和POLD3。FRAXA未能完成MiDAS会导致严重的基因座不稳定和有丝分裂的偏聚。我们建议断裂诱导的DNA复制是叶酸胁迫下FRAXA复制所必需的，并定义了人SLX1的细胞功能。这些发现提供了叶酸剥夺如何驱动人类基因组不稳定的见解。