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。存在几种称为检查点的机制，以确保在进入细胞周期的下一阶段之前，在细胞周期的特定时期内适当地执行事件。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。这些检查点的作用是暂时停止或减缓细胞周期，以纠正基因组完整性问题。检查点机制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。这些途径从酵母到人类都是保守的，这使得前者成为基本检查点研究的理想生物。两类蛋白激酶在信号转导中起关键作用。酵母菌Mec1激酶与ATR和ATM激酶有关，后者在共济失调毛细血管扩张症患者中存在缺陷；它的功能最接近DNA损伤部位和停滞的复制叉子。与人类Chk1和Chk2相关的Rad53蛋白激酶在下游起作用，阻止细胞周期并激活其他反应通路。这些蛋白激酶的催化中心在检查点进程中被激活。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起始蛋白Dpb11(在人类中称为TopBP1，在分裂酵母中称为Rad4/Cut5)激活Mec1，通过结构-功能分析研究Mec1激活的性质(Aim 2)，并研究DNA结合在Rad53激活中的作用(Aim 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。与人类细胞的公共健康相关，所谓的检查点在细胞分裂之前监测DNA的完整性，我们的遗传物质，以及DNA复制的正确性。检查点通路已知缺陷的患者患癌症的风险极高。检查点通路从人类到酵母都是保守的，我们建议在酵母中研究这些通路，因为作为模式生物，它更容易进行遗传和生化分析。

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. Several mechanisms, called checkpoints, exist to 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. These checkpoints function to temporarily halt or slow down the cell cycle in order to correct problems with genome integrity. Defects in the checkpoint mechanisms result in increased chromosome instability and such defects are known cancer predisposition conditions in human. These pathways have been conserved from yeast to human which makes the former an ideal organisms for basic checkpoint studies. Two classes of protein kinases are critical for signal transduction. The yeast Mec1 kinase is related to the ATR and ATM kinases, the latter being defective in ataxia telangiectasia patients; it functions most proximal to sites of DNA damage and to stalled replication forks. The Rad53 protein kinase, related to human Chk1 and Chk2, acts downstream in halting the cell cycle and activating other response pathways. The catalytic centers of these protein kinases are activated during checkpoint progression. 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 initiation protein Dpb11, called TopBP1 in human and Rad4/Cut5 in fission yeast (Aim 1), to study the nature of activation of Mec1 by structure-function analysis (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. PUBLIC HEALTH RELEVANCE In human cells, so-called checkpoints monitor the intactness of the DNA, our genetic material, and the correctness of DNA duplication prior to cell division. Patients with known defects in checkpoint pathways are at a highly increased risk for developing cancer. Checkpoint pathways are conserved from human to yeast, and we are proposing to study these pathways in yeast, because as a model organism it is more approachable to genetic and biochemical analysis.