An AT-rich sequence in human common fragile site FRA16D causes fork stalling and chromosome breakage in S-cerevisiae

Common fragile sites are regions of human chromosomes prone to breakage. Fragile site FRA16D spans the WWOX/FOR tumor suppressor gene and has been linked to cancer-causing deletions and translocations. Using a genetic assay in yeast, we found that a short AT-rich region (Flexl) within FRA16D increases chromosome fragility, whereas three other sequences within FRA16D do not. To our knowledge, this is the first identification of a sequence element within a common fragile site that increases chromosome fragility. The fragility of Flexl was exacerbated by the absence of Rad52 or the presence of hydroxyurea. Flexl contains a polymorphic AT repeat predicted to form a DNA structure, and two-dimensional gel analysis showed accumulation of stalled replication forks at the Flexl sequence that was dependent on AT length. Our data suggest that the FRA16D Flexl sequence causes increased chromosome breakage by forming secondary structures that stall replication fork progression.