Mitosis, the process whereby the duplicated genetic material is evenly segregated in two daughter cells, is a complex process that requires so called mitotic checkpoints to be properly orchestrated. Inactivation of these checkpoints has been reported in cancer cells and is thought to contribute to the genetic instability and aneuploidy that characterizes cancer. There are at least three mitotic checkpoints in normal human cells; one operates in prophase, one in metaphase (the spindle checkpoint) and the third regulates mitotic exit. Of these three checkpoints, the spindle checkpoint is the best studied, but it is not frequently inactivated in human cancer. The prophase checkpoint requires the activity of the Chfr gene, which is currently thought to be the most frequently mutated mitotic checkpoint gene in human cancer. Exit from mitosis requires the activity of several genes that comprise the mitotic exit network. Among them, the GTPase Teml activates the CdclS kinase, which in turn activates the Dbf2-LATSl/Mobl kinase. Analysis of mitotic checkpoint genes and their role in human cancer will require multiple experimental approaches. I propose to use a structure-based approach to characterize Chfr and the mitotic exit proteins Teml, CdclS, Dbf2-LATSl and Mobl. I have already solved the three-dimensional structure of the Chfr FHA domain and the structure of human Mobl A. Based on the Chfr structure, I hypothesize that the FHA domain of Chfr is involved in Chfr oligomerization and propose to test this hypothesis with a variety of structural, biochemical and molecular biology approaches. I also propose to solve the three-dimensional structure of the Teml-Cdcl5 complex, in order to understand the mechanism of CdclS activation. In addition, I propose to solve the crystal structure of a Mobl/Dbf2-LATSl complex in an effort to understand how Mob proteins regulate the activities of their associated kinases. These studies will help elucidate the function of genes that are important for mitotic progression and are inactivated in human cancer.
