Regulation and functions of DNA damage checkpoint kinases in Yeast

酵母 DNA 损伤检查点激酶的调控和功能

基本信息

批准号：
7779413
负责人：
HUILIN ZHOU
金额：
$ 28.57万
依托单位：
LUDWIG INSTITUTE FOR CANCER RES LTD
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-01 至 2012-03-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): A hallmark of cancer and other diseases are high rates of mutation and genome instability, which are believed to be the driving force for disease causing genetic changes. The DMA damage checkpoint is important for the suppression of mutations and genome instabilities, and the regulation of many cellular responses to DMA damage. While an increasing number of genes have been identified to function in the DMA damage checkpoint in human and mutations in some checkpoint genes are linked to cancers, fundamental questions of how the DNA damage checkpoint is regulated remain far from being understood. An important reason for using model experimental systems is to rapidly obtain experimental insights into biological problems that can be applied to problems of human diseases, especially for highly conserved pathways like the DNA damage checkpoint. Yeast, in particular, offers unparalleled advantages in yeast genetics and short generation time. Basic studies in yeast have contributed significantly to the understanding of the DNA damage checkpoint. In the proposed basic studies, we are primarily interested in two basic mechanistic questions about the DNA damage checkpoint. First, how is the DNA damage checkpoint activated? Second, how are various DNA damage checkpoint pathways linked to various DNA damage responses? Accordingly, there are three complementary aims in this proposal. First, we will examine the genetic pathways for Rad53 and Dun1 activation using SmM as a substrate. Second, we will dissect the role of Dun1 phosphorylation in its activation and characterize the roles of Dun1 phosphorylation in the functions of Dun1, including the regulation of dNTP levels, telomere maintenance and suppression of gross chromosome rearrangements. Third, we will use a newly developed Rad53- Dun1-Sml1 system to dissect how Mec1, Tell and Rad53 are activated via biochemical reconstitution and genetic analysis. We will use purified proteins to reconstitute the activation of the Rad53-Dun1-Sml1 system, and further use this system to study the regulation of Mec1 and Tell. Our long-term goal is to understand the regulation of the DNA damage checkpoint kinases and their functions in the regulation of cell growth and suppression of genome instability.

描述（由申请人提供）：癌症和其他疾病的标志是突变和基因组不稳定性的高率，这被认为是导致遗传变化的疾病的驱动力。 DMA损伤检查点对于抑制突变和基因组不稳定性以及许多细胞对DMA损伤的反应很重要。尽管已经确定了越来越多的基因在人类DMA损伤检查点的功能，并且某些检查点基因中的突变与癌症有关，但如何调节DNA损伤检查点的基本问题尚未被理解。使用模型实验系统的一个重要原因是，可以快速获得对可以应用于人类疾病问题的生物学问题的实验见解，尤其是对于高度保守的途径（例如DNA损伤检查点）。尤其是酵母菌，在酵母遗传学和短生成时间方面具有无与伦比的优势。酵母中的基础研究对DNA损伤检查点的理解做出了重大贡献。在拟议的基础研究中，我们主要对有关DNA损伤检查点的两个基本机械问题感兴趣。首先，如何激活DNA损伤检查点？其次，各种DNA损伤检查点途径如何与各种DNA损伤响应相关联？因此，该提案中有三个补充目标。首先，我们将使用SMM作为底物检查RAD53和DUN1激活的遗传途径。其次，我们将剖析DUN1磷酸化在其激活中的作用，并表征DUN1磷酸化在DUN1功能中的作用，包括调节DNTP水平，维持和抑制总染色体重排重排。第三，我们将使用新开发的RAD53-DUN1-SML1系统来剖析MEC1，Tell和Rad53如何通过生化重构和遗传分析激活。我们将使用纯化的蛋白质来重建RAD53-DUN1-SML1系统的激活，并进一步使用该系统来研究MEC1的调节并说明。我们的长期目标是了解DNA损伤检查点激酶的调节及其在调节细胞生长和抑制基因组不稳定性方面的功能。