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

许多人类癌症起源于DNA损伤反应和细胞周期控制的缺陷。这些缺陷导致了染色体的不稳定。为了鉴定与DNA损伤反应和细胞周期调控相关的新基因，我们使用了一种实验模式生物--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通路中发挥着重要作用。我们已经在线虫基因组中发现了一个类似ATM的Ce-Atl-1。RNA干扰(RNA1)使其基因产物缺失，导致染色体分离和DNA损伤反应的明显缺陷。酵母双杂交分析结果表明，其N端一半与增殖细胞核抗原相互作用，其C端一半与几个与内质网运输、内吞作用、转录因子、单链结合蛋白SSB相关的基因产物相互作用。…我们还分析了Skp1-CDC53/CUL1-F box(SCF)的常见亚基Ring-H2 Finger Protein RBX1，以及其他Cullins和von Hippel-Lindau(VHL)肿瘤抑制因子E3泛素连接酶复合体。RNAi实验表明，RBX1蛋白参与了与有丝分裂染色体凝聚和分离、G1/S进程和胞质分裂相关的多种功能。所有这些Rb×1RNAi缺陷都与CUL2、Elongin B(ELB1)或Elongin C(ELC1)缺失后发现的一致。此外，细菌双杂交分析还揭示了ELC1和CUL2之间以及ELC1和ELC2之间的直接相互作用。因此，RB×1/CUL2/ELC1/ELB1复合体在线虫中扮演着E3泛素连接酶的角色，是与染色体动力学和细胞周期控制相关的多种功能所必需的。这些现象可能在人类癌症中普遍存在，因为这项研究中发现的所有这些基因在哺乳动物中都高度保守。较少

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