Genetics of Cell Cycle Regulators in C.elegans

描述（由申请人提供）：细胞周期调控的中心目标是基因组DNA的忠实复制和DNA分离到两个子细胞。基因组DNA必须在每个细胞周期完全复制，但这种复制必须限于每个细胞周期的单轮。真核细胞广泛利用泛素介导的蛋白水解途径降解蛋白质来调节细胞周期转换。泛素连接酶促进底物蛋白的多聚泛素化，这标志着它们的降解。调节细胞周期的泛素连接酶的主要类别之一是cullin/RING指复合物。我们发现C.需要线虫cullin CUL-4将DNA复制限制在每个细胞周期一次。在不存在CUL-4的情况下，细胞停滞在S期并持续重新复制其基因组DNA。CUL-4限制基因组DNA复制的分子途径尚不清楚。相关的cullin蛋白CUL-1和CUL-2在具有专用底物结合组分的多蛋白cullin/RING指复合物中发挥作用。目前，CUL-4功能的蛋白复合物尚未在任何生物体中描述。C.线虫作为研究CUL-4功能的模式系统，可以利用强有力的遗传学和分子生物学手段，本研究的实验将阐明CUL-4的分子功能。Elegans CUL-4在三个方面。首先，免疫亲和和双杂交方法将用于鉴定在CUL-4复合物中起作用的蛋白质。其次，免疫亲和，双杂交，抑制筛选将用于确定CUL-4复合物的底物。这些CUL-4复合物组分和底物的分子和遗传表征有望为调节DNA复制起始的基本过程提供重要见解。最后，将确定CUL-4与真核DNA复制许可因子Cdc 6和Cdt 1的同源物之间的相互作用。除了提供对DNA复制的基本方面的见解外，这项工作还对理解癌症病因学具有影响，因为已发现人类CUL 4A在乳腺癌中过表达，并且涉及DNA再复制的癌基因的基因扩增是多种癌症的促成因素。

DESCRIPTION (provided by applicant): The central objective of cell cycle regulation is the faithful duplication of genomic DNA and the segregation of that DNA to two daughter cells. Genomic DNA must be replicated completely each cell cycle, yet this replication must be limited to a single round of per cell cycle. Protein degradation through the ubiquitin-mediated proteolytic pathway is employed extensively by eukaryotic cells to regulate cell cycle transitions. Ubiquitin ligases facilitate the polyubiquitination of substrate proteins, which marks them for degradation. One of the major classes of ubiquitin-ligases that regulate the cell cycle are cullin/RING finger complexes. We have found that the C. elegans cullin CUL-4 is required to limit DNA replication to once per cell cycle. In the absence of CUL-4, cells arrest in S phase and continuously re-replicate their genomic DNA. The molecular pathway through which CUL-4 functions to limit genomic DNA replication is not known. The related cullin proteins CUL-1 and CUL-2 function in multi-protein cullin/RING finger complexes with dedicated substrate-binding components. Currently the protein complex(es) in which CUL-4 functions have not been described in any organism. C. elegans is being used as a model system in which to study CUL-4 function, to allow the use of powerful genetic and molecular approaches.The experiments in this proposal will clarify the molecular functions of C. elegans CUL-4 in three ways. First, immunoaffinity and two-hybrid methods will be used to identify proteins that function in CUL-4-complexes. Second, immunoaffinity, two-hybrid, and suppressor screens will be used to identify substrates of CUL-4 complexes. The molecular and genetic characterization of these CUL-4 complex components and substrates is expected to provide significant insights into the fundamental processes that regulate the initiation of DNA replication. Finally, the interaction between CUL-4 and homologs of the eukaryotic DNA replication licensing factors Cdc6 and Cdt1 will be determined. In addition to providing insights into the fundamental aspects of DNA replication, this work has implications for understanding cancer etiology as human CUL4A has been found to be overexpressed in breast cancer, and gene amplification of oncogenes, which involves DNA re-replication, is a contributing factor in multiple cancers.