| An increase in DNA damage is an "enabling characteristic" in tumorigenesis, and normally occurs at loci called common fragile sites (CFSs), or by the loss of genes involved in the DNA damage response pathway. CFSs are a normal part of the human genome, and are exceptionally prone to DNA damage under conditions of replication stress, such as treatment with low concentrations of the DNA polymerase inhibitor aphidicolin. While much has been learned about the cellular responses to DNA damage at CFSs, less is known about what makes these sites inherently unstable. To determine if the sequence of CFSs is causal to their instability, BACs containing CFS sequence were stably transfected into HCT116 cells, and cell clones with BACs integrated at ectopic non-fragile loci were isolated. Cell clones containing integrated CFS BACs showed a significant increase in aphidicolin-induced DNA damage at the integration site, compared to control BACs. This is the first direct evidence in human cells that introduction of CFS sequences into ectopic non-fragile loci is sufficient to cause CFS-like instability. These data support the hypothesis that sequences at CFSs are inherently unstable, and are a major factor in the formation of replication stress induced gaps and breaks at CFSs.;A primary responder to DNA damage at CFSs, the protein kinase Ataxia-telangiectasia and rad3-related (ATR) is responsible for sensing and signaling DNA damage associated with stalled replication forks. The role of ATR and the maintenance of damage at CFSs, in tumorigenesis is not well understood due to the lethality of complete loss of ATR and is best studied in vivo. To overcome this limitation, we created a viable mouse model with a hypomorphic Atr mutation and an estimated 66-82% reduction in Atr protein levels. These mice exhibited increased APH-induced DNA damage and checkpoint abnormalities indicative of Atr deficiency. Creation of a mouse model with hypomorphic Atr expression will serve as a valuable reagent to study the biological effects of Atr deficiency in vivo. |
