KINASE ACTIVATION IN THE DNA DAMAGE CHECKPOINTS

描述（由申请人提供）：真核生物中的细胞周期是一个高度调节的过程网络，包含许多检查和平衡，以确保其正确和及时执行。细胞周期检查点确保细胞周期特定时期的事件在进入细胞周期的下一个阶段之前适当地进行。在这个提议中感兴趣的检查点机制是那些对DNA损伤和DNA复制过程中出现的问题做出反应的机制。检查点的缺陷导致染色体不稳定性增加，并且此类缺陷是人类中已知的癌症易感性状况。从酵母到人类，DNA损伤和DNA复制检查点途径都是保守的，这使得酵母成为基础检查点研究的理想生物。这项提案的主要重点将是传感器蛋白激酶Mec 1，人类ATR的直系同源物，启动检查点。Mec 1/ATR激酶活性作为检查点途径的起始步骤被激活，通过与显示细胞周期时相特异性的特异性激活剂相互作用。第二种蛋白激酶Rad 53与人Chk 1和Chk 2相关，在下游停止细胞周期并激活其他反应途径。虽然许多遗传和分子生物学研究已经确定了许多涉及各种检查点的因素，但Mec 1和Rad 53激活的生化机制仍然相对模糊。本提案中的生物化学研究旨在了解Mec 1通过复制蛋白Dna 2的激活（目的1）;通过冷冻电子显微镜和晶体学研究Mec 1的结构，并使用小肽作为探针通过结构-功能分析研究Mec 1激活的性质（目的2）;并研究DNA结合在Rad 53激活中的作用（目的3）。这些生化实验将通过遗传研究来补充，以获得检查点激活的综合观点。

DESCRIPTION (provided by applicant): The cell cycle in eukaryotic organisms is a highly regulated network of processes that contains numerous checks and balances to ensure its proper and timely execution. Cell cycle checkpoints ensure that the events during a specific period of the cell cycle are carried out appropriately before proceeding to the next phase of the cell cycle. The checkpoint mechanisms of interest in this proposal are those that respond to DNA damage and to problems arising during DNA replication. Defects in checkpoints result in increased chromosome instability and such defects are known cancer predisposition conditions in human. DNA damage and DNA replication checkpoint pathways have been conserved from yeast to human which makes the former an ideal organisms for fundamental checkpoint studies. The primary focus of this proposal will be on the sensor protein kinase Mec1, the ortholog of human ATR, that initiates the checkpoint. Mec1/ATR kinase activity is activated as the initiating step of the checkpoint pathway, through interaction with specific activators that show cell-cycle phase specificity. A second protein kinase, Rad53, which is related to human Chk1 and Chk2, acts downstream in halting the cell cycle and activating other response pathways. While numerous genetic and molecular biological studies have identified many of the factors involved in the various checkpoints, the biochemical mechanisms of activation of Mec1 and of Rad53 have remained relatively obscure. The biochemical studies in this proposal are aimed at understanding Mec1 activation by the replication protein Dna2 (Aim 1); to study the structure of Mec1 by cryo-electron microscopy and crystallogaphy and the nature of activation of Mec1 by structure-function analysis using small peptides as probes (Aim 2); and to study the role of DNA binding in activation of Rad53 (Aim 3). These biochemical experiments will be complemented by genetic studies to obtain an integrated view of checkpoint activation.