Common fragile sites: Mechanisms of instability.

Abstract

When DNA replication is perturbed, gaps and breaks appear on metaphase chromosomes at specific sites, called common fragile sites. Common fragile sites were first reported in human cells but are also present in other mammalian species. We have investigated several primates and found fragile sites in these species that are orthologous to human fragile sites, with the exception of gibbons, which lack an ortholog to FRA3B. Fragile site instability includes not only chromosomal gaps and breaks, but also translocations, deletions, foreign DNA integration and high frequency of SCE at these sites, both in cell culture and in cancer cells. Yet, the molecular mechanisms underlying fragile site instability have been elusive. We have investigated two key cell cycle checkpoint kinases, ATM and ATR, for their role in fragile site instability. We found that only ATR, which is known to respond to stalled replication fork progression, is crucial for preventing instability at fragile sites. We also investigated fragile sites in Seckel syndrome, a human disease resulting from a hypomorphic mutation in ATR, and found that cells from individuals with Seckel syndrome have significantly greater instability at fragile sites. Seckel syndrome is the first human disease to be associated with increased chromosome breakage at fragile sites. The ATR pathway is the first molecular pathway found to influence fragile sites and these results suggest that fragile sites represent unreplicated or single-stranded regions caused by replication stalling. We then investigated what type of repair processes may act at stalled or collapsed replication forks at fragile sites, beginning with homologous repair (HR) via cells lacking RAD51, an important HR protein. We determined that gaps and breaks and SCE at fragile sites are not affected by RAD51 deficiency. Thus, repair of replication forks at fragile sites and SCE formation at such sites is RAD51-independent. In summary, this dissertation research is important for understanding the reasons for fragile site instability, the consequences of stalled replication and its repair in normal cells, and the relevant mutations leading to fragile site instability in tumor cells and genetic disease.