Checkpoint protein interactions with a fragile site and chromosome instability

描述（由申请人提供）：我们研究的总体目标是了解检查点蛋白如何调节基因组稳定性。我们建议研究检查点蛋白和不稳定性产生在一个染色体位点，表现得像一个脆弱的位置。我们发现酵母脆弱位点的行为非常像哺乳动物的脆弱位点；酵母和哺乳动物的位点似乎都能阻止复制分叉，两者都能激活，然后通过与检查点蛋白的相互作用稳定下来。我们相信，酵母脆弱位点的研究提供了一个独特的机会来了解哺乳动物脆弱位点，以及研究在许多染色体位点上将停滞的复制叉与基因组不稳定联系起来的事件。我们已经开发了一个实验系统来研究这个位点，并认为它的不稳定性是由于DNA复制分叉的停滞和断裂，这在检查点突变体中大大增强。具体地说，我们提出这个位点的初始事件是tRNA基因停止DNA复制。第二，我们建议停滞的分叉崩溃或断裂。第三，我们提出DNA断裂与其他染色体位点（也可能是脆弱的）进行非等位基因重组以产生不稳定的易位。第四，我们认为不稳定易位之所以不稳定，是因为它有一个“超脆弱”的关节，容易发生额外的重排（不稳定循环）。最后，我们提出检查点和其他调节蛋白通过调控叉的失速、断裂和重组来影响位点的不稳定性。我们相信，了解这个位点上的每一个事件将有助于了解这些事件是如何在基因组的许多位点上发生的。这些研究的完成将有助于我们理解检查点蛋白如何维持基因组的稳定性。检查点是确保染色体正确复制的细胞控制。了解检查点是如何保持细胞基因组完整的，对于理解一个正常细胞如何变成癌细胞的过程至关重要。

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.