Enzymology of Mitosis Promoting Factor (MPF)

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Eukaryotic cells preserve the integrity of the genome by employing checkpoint surveillance systems. These regulatory networks detect the presence of incompletely replicated or damaged DNA in the nucleus and thereupon trigger a blockade to mitosis. In vertebrates, the process that detects incompletely replicated DNA, which is known as the DNA replication checkpoint, involves the upstream regulatory kinase ATR and the downstream effector kinase Chk1. In addition, there is a critical mediator protein called Claspin that regulates the activation of Chk1 by ATR. Claspin is a Chk1 -binding protein that associates directly with DNA replication forks and appears to be a sensor of ongoing DNA synthesis. Consequently, Claspin plays a pivotal role in the DNA replication checkpoint. The proposed studies will be aimed at understanding the function and regulation of Claspin. Most of these experiments will be conducted in Xenopus egg extracts, a system in which the components of the DNA replication checkpoint can be studied in a cell-free reaction. Specific experimental objectives will include studies of how modifications of Claspin and its protein-protein interactions control its ability to mediate the activation of Chk1. In conjunction with these studies, the molecular mechanism by which Claspin controls the activation of Chk1 will be explored. Significantly, it has been established that Claspin undergoes inactivation upon termination of a checkpoint response by a process called adaptation. The underlying mechanisms and broader physiological role(s) for the negative regulation of Claspin will be investigated. Finally, the hypothesis that Claspin is a multifunctional protein that possesses an additional, checkpoint-independent role that regulates the DNA replication fork will be explored. This research is relevant to public health because cancer arises from the progressive accumulation of mutations in key regulatory genes. Protein such as Claspin help to prevent the transmission of mutated genes at the time of cell division. Therefore, the proposed studies will aid in the understanding of how cells avoid cancer-inducing mutations.