Stably transfected common fragile site sequences exhibit instability at ectopic sites

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• Common fragile sites (CFSs) are chromosome loci susceptible to gaps and breaks under replication stress.
• The inherent instability of CFS sequences, rather than just cellular responses, is not well understood.
• CFS sequences are evolutionarily conserved and contain motifs potentially causing instability.

Purpose of the Study:

• To investigate the role of common fragile site (CFS) sequences in causing chromosome breakage.
• To determine if CFS sequences are inherently unstable and sufficient to induce fragility at ectopic sites.

Main Methods:

• Stable transfection of BACs containing FRA3B (CFS) or control sequences into HCT116 cells.
• Analysis of aphidicolin-induced gaps and breaks at BAC integration sites.
• Examination of replication timing at integrated CFS sequences.

Main Results:

• Integration of FRA3B BACs led to a 3-7 fold increase in aphidicolin-induced gaps and breaks.
• FRA3B integration sites exhibited chromosome rearrangements characteristic of CFS instability.
• Fragility of integrated FRA3B sequences was independent of late replication timing.

Conclusions:

• CFS sequences are inherently unstable and sufficient to cause fragility when integrated into ectopic loci.
• Sequence-intrinsic properties of CFSs are a major factor in replication stress-induced chromosome instability.
• This study provides direct evidence in human cells supporting the inherent instability hypothesis of CFSs.