Folate stress induces SLX1- and RAD51-dependent mitotic DNA synthesis at the fragile X locus in human cells

Abstract

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.

The RAD51 recombinase protects mitotic chromatin in human cells

“…It is, nonetheless, noteworthy that, while -H2AX-associated MiDAS was not significantly affected by the K133R mutation, B02-mediated mitotic RAD51 inhibition clearly reduced it (Figure 2I). Indeed, during the preparation of this manuscript, RAD51 was shown to catalyse break-associated MiDAS at the Fragile X locus, a folate-sensitive rare fragile site (Garribba et al, 2020). Therefore, while BIR-like MiDAS is also RAD51-dependent, it is surprising that the K133R mutation, which affects both the HR and protective functions of RAD51, does not appear to affect break-associated MiDAS.…”

Under-Replicated DNA: The Byproduct of Large Genomes?

“…Pif1 is the main helicase for yeast BIR [ 78 ]; however, the identity of the BIR and MiDAS helicase in human cells remains elusive. It is also important to address the universality of MiDAS, as it was shown that (i) folate stress induces SLX1- and RAD51-dependent (but RAD52 and MUS81 independent) mitotic DNA synthesis at rare fragile sites in human cells [ 79 ]; (ii) in non-cancerous human cell lines, APH-induced mitotic DNA synthesis is mainly FANCD2-dependent while RAD52 seems to be dispensable for this process [ 80 ].…”

“…The Lukas lab shed some light on this question by showing that a percentage of 53BP1-NBs are resolved in the next S phase through a RAD52-dependent replication-coupled repair mechanism (see Section 8.2 ) [ 96 ]. New data from the cell cycle field added another layer of complexity: the amount of inherited DNA damage has a direct impact on the immediate fate of daughter cells [ 79 , 80 , 81 ]. After each mitosis, actively proliferating cells assess not only the availability of mitogens but also their ‘genome health’ (unresolved lesions still present) and then decide whether to continue proliferating (S phase entry commitment) or to activate a p21-dependent transient cell cycle exit (quiescence/G0 state) [ 109 , 110 , 111 ].…”

Folate Deficiency Triggers the Abnormal Segregation of a Region With Large Cluster of CG-Rich Trinucleotide Repeats on Human Chromosome 2

“…Next, we asked whether MiDAS, which serves as a rescue pathway to complete locus duplication in mitosis, and is known to occur at CFSs ( Minocherhomji et al, 2015 ), telomeres ( Minocherhomji et al, 2015 ; Dilley et al, 2016 ; Min et al, 2017 ; Ozer et al, 2018 ) and FRAXA ( Garribba et al, 2020 ), could occur at FOLD1 following folate stress treatment. To address this, we investigated whether mitotic EdU incorporation could be observed at the sites of bending FOLD1 following folate stress using an established protocol ( Garribba et al, 2018 ) in GM09237 cells ( Supplementary Figure 3 ).…”

“…Based on the previous knowledge of fragility and missegregation at the abnormally expanded FRAXA locus under folate stress ( Santoro et al, 2012 ; Bjerregaard et al, 2018 ; Garribba et al, 2020 ), we investigated whether folate deficiency could perturb other genomic regions containing CG-rich repeat sequences. To address this, we analyzed the stability of the FOLD1 region on Chr2, which contains the largest cluster of CG-rich TNRs present in the human genome under folate stress conditions.…”