In eukaryotes, cyclin-dependent kinases (CDKs) control critical steps in both the cell division cycle and the transcription cycle of RNA polymerase II. CDK7 is unique among metazoan CDKs in that it performs essential functions in both cycles, as the major CDK-activating kinase (CAK) and as a component of transcription factor IIH (TFIIH). CDK7 is therefore the central element in a network linking cell division with gene expression--a connection that may be disrupted in cancer. Another hallmark of cancer cells is the dysfunction of surveillance mechanisms that insure fidelity of genome duplication and segregation; CDK7 and related enzymes in fungi have been implicated both in the repair of, and in the cell-cycle response to, DNA damage. Thus, understanding how the CAK network coordinates cell division, growth and the DNA damage response is crucial, both for understanding how that control is lost in cancer, and for targeting the pathway in anti-cancer therapy. It has been difficult to discern how CDK7 is regulated, however, precisely because of its many required functions. It is clear that the different catalytic functions of CDK7 can be independently regulated; the proposed work aims to uncover the regulators and to discover new targets for CDK7 action.
The first aim i s to identify signals and signaling molecules that affect the CAK function of CDK7, by enzymologic characterization of different CDK7-containing complexes and by investigating the role of phosphorylation in determining CDK7 subcellular localization.
The second aim i s to test the hypothesis that CDK7 is a regulator of key cell cycle transitions and/or specific transcriptional programs by both classical- and chemical-genetic manipulation of CDK7 activity in vivo and in vitro.
The third aim i s to identify and characterize the enzymes that modulate CDK7's phosphorylation state, and thereby its stability and catalytic power, by classical biochemical methods. Finally, understanding of the CAK network will be expanded by characterization of a novel mammalian CDK-like kinase related to Csk1, a key component of the yeast CAK network. The mammalian kinase can substitute for Csk1 in yeast to execute a critical function in the DNA damage response. That it plays a similar role in mammalian cells exposed to DNA-damaging agents will be tested by ablating its function.
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