AT-Dinucleotide Rich Sequences Drive Fragile Site Formation

Common fragile sites (CFSs) are genomic regions prone to breakage under replication stress conditions. These sites are recurrently rearranged in cancer, contributing to oncogenesis by driving general genome instability. We have previously found that many CFSs are highly enriched with AT-dinucleotide rich sequences (AT-DRSs), which have the potential to form secondary structures that can impede the progression of DNA replication forks. Recently, we have demonstrated that AT-DRSs at the CFS FRA16C can arrest the progression of replication forks, perturbing the replication dynamics in this region. In the present work we investigated the direct role of AT-DRSs in chromosomal instability at CFSs. For this, a 3.4 kb long AT-DRS, derived from the CFS FRA16C, was introduced into an ectopic non-fragile locus on chromosome X in the human fibrosarcoma cell line HT1080. Cytogenetic analysis of metaphase chromosomes of cells grown under replication stress conditions revealed gaps and breaks at the integrated AT-DRS in 3.4% of the X chromosomes, indicating the ability of this sequence to form a novel fragile site. Moreover, sequences derived from the 3.4 kb long AT-DRS demonstrated an increased tendency to form stable secondary structures in vitro relative to control sequences. The ability of the 3.4 kb long AT-DRS to drive chromosomal instability under replication stress conditions indicates that AT-DRSs are a major factor underlying the molecular basis for fragility and emphasizes that intrinsic DNA features affecting DNA replication predispose the human genome to instability.