KINASE ACTIVATION IN THE DNA DAMAGE CHECKPOINTS

描述(申请人提供)：真核生物中的细胞周期是一个高度受监管的过程网络，其中包含许多制衡，以确保其正确和及时的执行。细胞周期检查点确保在进入细胞周期的下一阶段之前，适当地执行细胞周期特定时期内的事件。这项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。检查点的缺陷会导致染色体的不稳定性增加，这种缺陷是已知的人类癌症易感条件。DNA损伤和DNA复制检查点通路从酵母到人类都是保守的，这使得前者成为基础检查点研究的理想生物。这项提案的主要焦点将是启动检查点的传感器蛋白激酶Mec1，它是人类ATR的同源基因。Mec1/ATR激酶活性作为检查点通路的起始步骤，通过与表现出细胞周期时相特异性的特定激活剂相互作用而被激活。另一种与人类Chk1和Chk2相关的蛋白激酶Rad53在下游作用，阻止细胞周期并激活其他反应途径。虽然大量的遗传学和分子生物学研究已经确定了许多与不同检查点有关的因素，但Mec1和Rad53激活的生化机制仍然相对不明。本建议中的生化研究旨在了解复制蛋白DNA2(目标1)对Mec1的激活；用冷冻电子显微镜和晶体学研究Mec1的结构，以及以小肽为探针通过结构-功能分析研究Mec1的激活性质(目标2)；以及研究DNA结合在Rad53激活中的作用(目标3)。这些生化实验将得到基因研究的补充，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.