Checkpoint protein interactions with a fragile site and chromosome instability

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DESCRIPTION (provided by applicant): The overall goal of our research is to understand how checkpoint proteins regulate genome stability. We propose to study checkpoint proteins and instability arising at one chromosomal site that behaves like a fragile site. We found that the yeast fragile site behaves very much like a mammalian fragile site; both yeast and mammalian sites appear to stall replication forks, and both activate and are then stabilized by interaction with checkpoint proteins. We believe that study of the yeast fragile site provides a unique opportunity to understand mammalian fragile sites as well as to study events that link stalled replication forks to genome instability at many chromosomal sites. We have developed an experimental system to study this site, and believe its instability is due to stalling and breakage of DNA replication forks that is greatly enhanced in checkpoint mutants. Specifically, we propose that the initial event at this site is the stalling of DNA replication by tRNA genes. Second, we propose that the stalled forks collapse or break. Third, we propose that DNA breaks undergo non-allelic recombination with other chromosomal sites (that may also be fragile) to generate unstable translocations. Fourth, we propose the unstable translocation is unstable because it has a "hyperfragile" joint prone to additional rearrangements (cycles of instability). Finally, we propose that checkpoint and other regulatory proteins regulate fork stalling, breakage, and recombination to influence the sites instability. We believe that understanding each of these events at this site will be informative for understanding how such events occur at many sites in the genome. Completion of these studies will contribute to our understanding of how checkpoint proteins maintain genome stability. Checkpoints are cellular controls that ensure that chromosomes are correctly duplicated. Understanding how checkpoints keep a cell's genome intact is critical to understanding the process of how a normal cell becomes a cancer cell.