The sporatic or familial loss of checkpoint genes is associated with many types of cancer, especially lymphoma and leukemia. ATM, and the related checkpoint gene ATR, as well as the 9-1-1 """"""""PCNA-like"""""""" checkpoint genes are highly conserved from humans to yeast. We have generated GFP fusions to yeast checkpoint genes, allowing us to observe recruitment of checkpoint proteins to a defined enzymatically induced double stranded DNA break in vivo. We will use these reagents in several ways. First, we will examine the loading of each checkpoint complex onto damaged chromosomes during checkpoint activation and the removal of these proteins when the checkpoint pathway is turned off during checkpoint adaptation. Second, we will use these tools to examine the behavior of broken chromosomes during mitosis. Our data suggests that the broken ends of chromosomes remain associated. We will examine the behavior of broken chromosomes both during a checkpoint arrest and after cells have adapted to the checkpoint and carried the broken chromosome through mitosis. Our previous work has shown that some forms of genomic instability, such as chromosomal translocations, only occur after broken chromosomes are segregated after checkpoint adaptation.
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