Common Fragile Sites Are Characterized by Faulty Condensin Loading after Replication Stress

Abstract

Summary Cells coordinate interphase-to-mitosis transition, but recurrent cytogenetic lesions appear at common fragile sites (CFSs), termed CFS expression, in a tissue-specific manner after replication stress, marking regions of instability in cancer. Despite such a distinct defect, no model fully provides a molecular explanation for CFSs. We show that CFSs are characterized by impaired chromatin folding, manifesting as disrupted mitotic structures visible with molecular fluorescence in situ hybridization (FISH) probes in the presence and absence of replication stress. Chromosome condensation assays reveal that compaction-resistant chromatin lesions persist at CFSs throughout the cell cycle and mitosis. Cytogenetic and molecular lesions are marked by faulty condensin loading at CFSs, a defect in condensin-I-mediated compaction, and are coincident with mitotic DNA synthesis (MIDAS). This model suggests that, in conditions of exogenous replication stress, aberrant condensin loading leads to molecular defects and CFS expression, concomitantly providing an environment for MIDAS, which, if not resolved, results in chromosome instability.

Unscheduled origin building in S-phase upon tight CDK1 inhibition suppresses CFS instability

“…Further analyses showed that stimulation of replication in ARO-treated cells concerns all SDRs and T-SDRs, resulting in drastic reduction of windows with URI≤-2 (Figures 2C, 3 and S2F). These results indicate that inhibition of CDK1 by RO stimulates replication of CFSs, which explains their stability in cells grown in ARO, and also exclude the recently proposed hypothesis that G2 phase lengthening passively permits completion of CFS replication 42 .…”

“…This phenomenon, called mitotic DNA synthesis (MIDAS), was however considerably attenuated or totally absent in cells closer to normal. We propose that, in primary fibroblasts such as HS68 49 and MRC5 (this work) as well as in immortalized HBE bronchial epithelial cells 50 , RPE1 retina epithelial cells 42 and lymphoblasts (this work), CDK1 inhibition stimulates replication completion of CFSs during the S phase while some cell lines derived from advanced tumors are deficient in this DRC response. Altogether, results suggest that MIDAS is used as a backup mechanism in cells that fail to rescue CFS replication during the S phase, and that such failure strongly contributes to generate the genomic alterations found in cells of various cancers.…”

“…In striking contrast, it was recently reported that replication completion of CFSs occurs during mitosis in cancer-derived U2OS, HeLa and HCT116 cells grown in the very same conditions, i.e . for 16-24 h in ARO 42, 49, 50 . This phenomenon, called mitotic DNA synthesis (MIDAS), was however considerably attenuated or totally absent in cells closer to normal.…”

Mind the replication gap

“…DNA (re)synthesis is subsequently carried out by a POLD3-containing polymerase Pol δ . However, a recent study shows that depletion of either condensin I or condensin II, the essential complexes for chromosome condensation, does not impair, but increases MiDAS [ 83 ]. Therefore, whether DNA compaction is the trigger for MiDAS remains elusive.…”

SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication

“…Recent studies also highlight numerous “difficult-to-replicate” regions (Cortez, 2015; Gadaleta and Noguchi, 2017), including DNA-protein complexes, repetitive DNA such as centromeres and telomeres, and secondary DNA structures. Replicating such regions requires support by specific proteins, without which replication tends to fail, leading to genome instability (Cortez, 2015; Gadaleta and Noguchi, 2017) and the formation of fragile sites (Boteva et al, 2020). These observations highlight the importance of modulating chromatin structure during the replication process.…”