Co-treatment with low concentrations of the topoisomerase I inhibitor, camptothecin (CPT), reduces APH-induced breakage. CFS regions are highly conserved in mouse and other species, including primates, cat, dog, pig, horse, cow, Indian mole rat, and yeast (for review, see ). While CFSs could be a result of higher-order chromosome structure, the conservation throughout species could also indicate that they may have some conserved biological purpose. Mechanisms of instability The instability of CFSs is proposed to stem from late replication: CFSs are likely to initiate proper replication but slow to complete it, introducing breaks from unreplicated regions of DNA. Late-replication may be a result of formation of non-B DNA structures like hairpins and toroids that stall the replication fork in AT rich regions, analogous to the proposed mechanism of rare fragile site instability. Ataxia-telengiectasia and Rad3 Related (ATR) checkpoint kinase is required for maintaining stability of CFS under both stressed and normal replicating conditions. Breakage is reduced after treatment with CPT (camptothecin) (without APH), signifying that CPT also has a necessary role in stabilizing CFSs. Clinical relevance Fragile sites are associated with numerous disorders and diseases, both heritable and not. The FRAXA site is perhaps most famous for its role in Fragile X syndrome, but fragile sites are clinically implicated in many other important diseases, such as cancer. FRA3B and FRA16D lie within the large tumor-suppressor genes, FHIT and WWOX, respectively. High frequency of deletions at breakpoints within these fragile sites has been associated with many cancers, including breast, lung, and gastric cancers (for review, see ) MicroRNA genes, which are preferentially involved in chromosomal alterations, are frequently located at fragile sites. Chromosomal alterations may lead to deregulation of microRNA, which could be of diagnostic and prognostic significance for cancers. Additionally, the Hepatitis B virus (HBV) and HPV-16 virus, the strain of human papilloma virus most likely to produce cancer, appear to integrate preferentially in or around fragile sites, and it has been proposed that this is crucial to the development of tumors. Fragile sites have also been implicated in a variety of syndromes (for a review, see ). For example, breakage at or near the FRA11b locus has been implicated in Jacobsen syndrome, which is characterized by loss of part of the long arm of chromosome 11 accompanied by mild mental retardation. The FRAXE site is associated in the development of a form of mental retardation without any distinctive phenotypic