G2/M DNA DAMAGE CHECKPOINT IN BUDDING YEAST

芽殖酵母中的 G2/M DNA 损伤检查点

基本信息

批准号：
6125409
负责人：
TED A. WEINERT
金额：
$ 21.88万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-01-01 至 2001-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6125409
关键词：
DNA binding protein DNA damage DNA repair Saccharomyces cerevisiae cell cycle fungal genetics gene expression gene mutation immunofluorescence technique protein protein interaction protein purification site directed mutagenesis temperature sensitive mutant yeast two hybrid system

项目摘要

DESCRIPTION: In eukaryotic cells, DNA damage causes arrest of the cell cycle to allow repair of this damage. A number of genes, termed "checkpoint" genes, required for this arrest have been identified in yeast and in other organisms. Human checkpoint genes include p53 and ATM and mutations in these genes are associated with increased probability of developing cancer. Dr. Weinert suggests that there may be three types of checkpoint proteins: 1) sensor proteins which bind and, perhaps, process DNA damage, 2) signal proteins that interact with the sensor protein and transduce a signal to target genes, and 3) the target genes, which directly mediate cell cycle arrest. His first series of experiments concern how sensor proteins act on DNA damage. Strains with the cdc13 mutation develop long single-stranded regions near the end of the chromosome. Dr. Weinert has found that mutations in the rad24 checkpoint gene reduced this degradation, and mutations in the rad9 checkpoint gene increased the degradation. He will determine whether the degradation occurs 3' to 5' or 5' to 3', and he will examine the effects of other mutations (for example, rad17) on the degradation process. The Rad17p shares homology with Rec1p, a known 3' to 5' exonuclease. Dr. Weinert will purify Rad17p and determine whether Rad17p is a 3' to 5' exonuclease. He will also try to localize Rad17p, Rad24 and Mec3p to sites of meiosis-specific DSB's using immunofluorescence. The point of these studies is to test the hypothesis that these proteins represent sensor proteins that process the cdc13-caused DNA damage. The second part of the proposal is designed to address two questions. First, is processing of DNA damage by Rad24p and Rad17p required to repair this DNA damage? Second, does cell cycle arrest require Rad24p and Rad17p to process the DNA damage? Dr. Weinert will try to answer these questions by mutating RAD17 and RAD24, and determining whether any of the mutant alleles show separation of function. For example, if he obtains a rad17 mutant that does not process DNA damage, but does show cell-cycle arrest, he will have demonstrated that DNA processing is not required for cell-cycle arrest. The third part of the proposal concerns new checkpoint mutants. His previous hunts involved looking for mutants that had poor viability after exposure to DNA damage. His new hunt will employ a direct search for cells that fail to show G2/M arrest in response to DNA damage. He hopes that this search will identify target genes of the checkpoint response. The last series of experiments investigate the order of function of genes in the checkpoint pathway. Dr. Weinert proposes looking at protein-protein interactions between different checkpoint proteins using the two-hybrid system or affinity columns. He also proposes two types of epistasis tests. Work from the Elledge lab suggests that the Rad53p is phosphorylated as part of the checkpoint response and functions downstream of Mec1p and the sensor proteins. Dr. Weinert will attempt to confirm this conclusion. If the conclusion is confirmed, he will determine the effect of various checkpoint mutations on the phosphorylation of Rad53p. He will also attempt to isolate a mec1 mutant with the phenotype of cold-sensitive constitutive function. He will then examine interactions with other mutants. A double mutant with a mec1 constitutive and a mutant in a gene that functions downstream of MEC1 should fail to show arrest at the restrictive temperature.

描述：在真核细胞中，DNA损伤导致细胞停滞循环以修复此损坏。许多基因称为在酵母中已经确定了此逮捕所需的“检查点”基因和其他生物。人类检查点基因包括p53和atm，以及这些基因中的突变与增加的概率有关发展癌症。 Weinert博士建议可能有三种类型的检查点蛋白质：1）结合并可能处理的传感器蛋白 DNA损伤，2）与传感器蛋白相互作用的信号蛋白和将信号转换为靶基因，3）靶基因，直接介导细胞周期停滞。他的第一个系列实验涉及传感器蛋白如何作用于DNA 损害。 Cdc13突变的菌株会形成长单链染色体尽头的区域。 Weinert博士发现 RAD24检查点基因中的突变减少了这种降解，并且 RAD9检查点基因中的突变增加了降解。他会的确定降解是否发生3'至5'还是5'至3'，他将检查其他突变（例如Rad17）对退化过程。 RAD17P与REC1P共享同源性，这是已知的3'至 5'外切酶。 Weinert博士将净化RAD17P并确定RAD17P是否是3'至5'的外切酶。他还将尝试定位Rad17p，Rad24和 MEC3P使用免疫荧光到减数分裂特异性DSB的位点。这这些研究的目的是检验这些蛋白质的假设表示处理CDC13引起的DNA损伤的传感器蛋白。该提案的第二部分旨在解决两个问题。首先，是修复需要的RAD24P和RAD17P的DNA损伤处理这种DNA损伤？其次，细胞周期停滞需要rad24p和rad17p 处理DNA损伤？ Weinert博士将尝试回答这些问题通过突变rad17和rad24，并确定是否有任何突变体等位基因显示功能分离。例如，如果他获得了Rad17 不处理DNA损伤但确实显示细胞周期停滞的突变体，他将证明细胞周期不需要DNA处理逮捕。该提案的第三部分涉及新的检查点突变体。他的以前的狩猎涉及寻找在此之后生存能力差的突变体暴露于DNA损伤。他的新狩猎将直接搜索细胞由于响应DNA损伤，这种情况无法显示G2/M停滞。他希望这个搜索将识别检查点响应的目标基因。最后一系列实验研究了基因在中的功能顺序检查点途径。 Weinert博士提议查看蛋白质蛋白质使用两个杂交的不同检查点蛋白之间的相互作用系统或亲和力列。他还提出了两种类型的上毒测试。来自Eledge Lab的工作表明Rad53p是磷酸化的一部分 MEC1P和传感器下游的检查点响应和功能蛋白质。 Weinert博士将试图确认这一结论。如果是结论已确认，他将确定各种检查站的效果 RAD53P磷酸化的突变。他还将尝试隔离具有冷敏感性功能表型的MEC1突变体。然后，他将检查与其他突变体的相互作用。带有双重突变体 MEC1的MEC1组成型和一个突变体，该基因在Mec1下游起作用应该在限制性温度下显示停滞。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Details and concerns regarding the G2/M DNA damage checkpoint in budding yeast.

有关芽殖酵母中 G2/M DNA 损伤检查点的详细信息和担忧。

DOI：
10.1101/sqb.2000.65.433
发表时间：
2000
期刊：
Cold Spring Harbor symposia on quantitative biology
影响因子：
0
作者：
Weinert,T;Little,E;Shanks,L;Admire,A;Gardner,R;Putnam,C;Michelson,R;Nyberg,K;Sundareshan,P
通讯作者：
Sundareshan,P

Use of cdc13-1-induced DNA damage to study effects of checkpoint genes on DNA damage processing.

利用 cdc13-1 诱导的 DNA 损伤来研究检查点基因对 DNA 损伤处理的影响。