Studies of the DNA Replication Checkpoint

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DESCRIPTION (provided by applicant): The challenging task of replicating a eukaryotic genome is made more difficult by intrinsic and extrinsic agents that interrupt DNA replication. To cope with these difficulties, cells have a DNA replication monitoring system that senses stalled replication forks and direct various responses. One of these responses is the S-M checkpoint that delays the onset of mitosis (M) while DNA synthesis (S) is underway. Other responses control DNA replication, repair and recombination. These responses are collectively termed the replication checkpoint. This application proposes to use the fission yeast Schizosaccharomyces pombe to investigate the replication checkpoint. Fission yeast has played a pioneering role in the discovery and analysis of checkpoint mechanisms that are conserved amongst all eukaryotes, including humans. The studies will focus on he protein kinase Cds1, a major effector of the checkpoint. One aim is to understand how Cds1 is regulated. These studies will concentrate on Mrc1, a protein then channels the replication checkpoint signal from the sensor kinase Rad3 to Cds1. The second aim is to understand how stalled replication forks are stabilized. Proteins involved in this process I will be identified and analyzed. The third aim is to characterize Cds1 targets. These studies will focus on Mus81 and Eme1, components of a Holliday junction resolvase that cleaves X-structured DNA. All of these genes have confirmed or presumptive homologs in humans. Some of these homologs are known to be important for maintaining genome integrity and preventing cancer, but how they accomplish this task is largely unknown. It is anticipated that studies of the replication checkpoint in fission yeast will provide a valuable framework for understanding replication checkpoint control in humans.