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Characterization ofgenes involved in genomic stability using an experinental model organism.

使用实验模型生物体表征参与基因组稳定性的基因。

基本信息

批准号：
13214008
负责人：
HIGASHITANI Atsushi
金额：
$ 20.03万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

项目摘要

Many human cancers originate from defects in the DNA damage response and the cell cycle control. These defects cause instability of chromosomes. To identify and characterize novel genes of DNA damage response and the cell cycle control, we used an experimental model organism the nematode Caenorthabditis elegans.In eukaryotic cells, members of the ataxia telangiectasia mutated gene (ATM) family play important roles in conserved signal transduction pathways for the DNA damage response. We have identified an ATM-like Ce-atl-1 in the C. elegans genome. Depletion of its gene product by RNA-mediated interference (RNA1) caused pronounced defects in chromosomal segregation and DNA damage responses such as repair, apoptosis, and G1 arrest From the results of yeast two-hybrid analysis, its N-terminal half physically interacted with PCNA and its C-terminal half interacted with several gene products related to transport of ER, endocytosis, transcriptional factor, single strand binding protein SSB. … More Some of them were essential for apoptosis, but dispensable for DNA repair process.We also analyzed the Ring-H2 finger protein RBX1 that is a common subunit of SKP1-CDC53/CUL1-F box (SCF), other Cullins and von Hippel-Lindau (VHL) tumour suppressor E3 ubiquitin ligase complexes. The RBX1 RNAi experiment suggested that the RBX1 protein participates in diverse functions relevant to mitotic chromosomal condensation and segregation, G1/S progression and cytokinesis. All these defects of RB×1 RNAi were consistent with those found after either depletion of CUL2, Elongin B (ELB1) or Elongin C (ELC1). In addition, direct interactions between ELC1 and CUL2, and ELC1 and ELC2, respectively, were revealed by bacterial two-hybrid analysis. Thus, the RB×1/CUL2/ELC1/ELB1 complex acts as an E3 ubiquitin ligase in C. elegans and is essential for diverse functions relevant to chromosomal dynamics and cell cycle control. These phenomena are probably ubiquitous in human cancer, because all these genes identified in this study are highly conserved in mammals. Less

许多人类癌症起源于DNA损伤反应和细胞周期控制的缺陷。这些缺陷导致染色体不稳定。为了鉴定和表征DNA损伤反应和细胞周期控制的新基因，我们使用了一种实验模式生物——秀丽隐杆线虫。在真核细胞中，共济失调毛细血管扩张突变基因（ATM）家族成员在DNA损伤反应的保守信号转导途径中发挥重要作用。我们已经在秀丽隐杆线虫的基因组中发现了一个类似于atm的Ce-atl-1。其基因产物被rna介导的干扰（RNA1）耗尽导致染色体分离和DNA损伤反应（如修复、凋亡和G1阻滞）明显缺陷。从酵母双杂交分析结果来看，其n端一半与PCNA物理相互作用，其c端一半与ER转运、内吞噬、转录因子、单链结合蛋白SSB相关的几个基因产物相互作用。其中一些是细胞凋亡所必需的，但在DNA修复过程中是必不可少的。我们还分析了SKP1-CDC53/CUL1-F box （SCF）、其他Cullins和von Hippel-Lindau （VHL）肿瘤抑制因子E3泛素连接酶复合物的常见亚基Ring-H2指蛋白RBX1。RBX1 RNAi实验表明，RBX1蛋白参与了与有丝分裂染色体凝聚分离、G1/S进程和细胞分裂相关的多种功能。RB×1 RNAi的这些缺陷与CUL2、伸长蛋白B （ELB1）或伸长蛋白C （ELC1）缺失后发现的缺陷一致。此外，通过细菌双杂交分析，揭示了ELC1与CUL2、ELC1与ELC2之间的直接相互作用。因此，RB×1/CUL2/ELC1/ELB1复合物在秀丽隐杆线虫中作为E3泛素连接酶，对染色体动力学和细胞周期控制相关的多种功能至关重要。这些现象可能在人类癌症中普遍存在，因为本研究中发现的所有这些基因在哺乳动物中都是高度保守的。少