The CAK Network in Metazoan Cell Cycle Regulation

超出所提供的空间。在真核生物中，细胞周期蛋白依赖性激酶（CDK）控制细胞分裂周期和RNA聚合酶II转录周期中的关键步骤。CDK 7在后生动物CDK中是独特的，因为它在两个周期中都发挥重要作用，作为主要的CDK激活激酶（CAK）和转录因子IIH（TFIIH）的组分。因此，CDK 7是连接细胞分裂与基因表达的网络中的中心元件-这种连接可能在癌症中被破坏。癌细胞的另一个标志是确保基因组复制和分离的保真度的监视机制的功能障碍;真菌中的CDK 7和相关酶涉及DNA损伤的修复和对DNA损伤的细胞周期反应。因此，了解CAK网络如何协调细胞分裂，生长和DNA损伤反应至关重要，这对于了解癌症中的控制如何丢失以及抗癌治疗中的靶向途径都至关重要。然而，很难辨别CDK 7是如何调节的，这正是因为它有许多必需的功能。很明显，CDK 7的不同催化功能可以独立调节;拟议的工作旨在揭示调节器并发现CDK 7作用的新靶点。第一个目的是通过对不同的含CDK 7的复合物进行酶学表征，并通过研究磷酸化在确定CDK 7亚细胞定位中的作用，来鉴定影响CDK 7的CAK功能的信号和信号分子。第二个目的是通过体内和体外CDK 7活性的经典和化学遗传操作来检验CDK 7是关键细胞周期转换和/或特定转录程序的调节剂的假设。第三个目的是通过经典的生物化学方法来鉴定和表征调节CDK 7磷酸化状态的酶，从而确定其稳定性和催化能力。最后，CAK网络的理解将扩大一个新的哺乳动物CDK样激酶相关的Cskl，酵母CAK网络的关键组成部分的表征。哺乳动物激酶可以替代酵母中的Cskl以在DNA损伤反应中执行关键功能。它在暴露于DNA损伤剂的哺乳动物细胞中起着类似的作用，这将通过消除其功能来测试。性能现场=

EXCEED THE SPACE PROVIDED. 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 is to identify signals and signaling molecules that affect the CAK function of CDK7, by enzymologic characterization of different CDK7-containing complexes, and by linvestigating the role of phosphorylation in determining CDK7 subcellular localization. The second aim is 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 is 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 Cskl, a key component of the yeast CAK network. The mammalian kinase can substitute for Cskl 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. PERFORMANCE SITE ========================================Section End===========================================