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。存在几种称为检查点的机制，以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。这些检查点的功能是暂时停止或减缓细胞周期，以纠正基因组完整性的问题。检查点机制中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。这些途径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体。两类蛋白激酶对信号转导至关重要。酵母Mec 1激酶与ATR和ATM激酶相关，后者在共济失调毛细血管扩张症患者中有缺陷;它的功能最接近DNA损伤位点和停滞的复制叉。Rad 53蛋白激酶与人Chk 1和Chk 2相关，在下游起作用，停止细胞周期并激活其他反应途径。这些蛋白激酶的催化中心在检查点进展期间被激活。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被复制起始蛋白Dpb 11（在人类中称为TopBP 1，在裂变酵母中称为Rad 4/Cut 5）激活（Aim 1），通过结构-功能分析研究Mec 1激活的性质（Aim 2），并研究DNA结合在Rad 53激活中的作用（Aim 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。 在人类细胞中，所谓的检查点监测DNA的完整性，我们的遗传物质，以及细胞分裂前DNA复制的正确性。检查点通路中存在已知缺陷的患者患癌症的风险极高。从人类到酵母，检查点途径是保守的，我们建议在酵母中研究这些途径，因为作为一种模式生物，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.