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的忠实复制和该DNA分离到两个子细胞。基因组DNA必须在每个细胞周期内完全复制，但这种复制必须限制在每个细胞周期的单个周期内。真核细胞广泛利用泛素介导的蛋白水解途径来调节细胞周期转变。泛素连接酶促进底物蛋白的多泛素化，这标志着它们的降解。调控细胞周期的主要泛素连接酶之一是cullin/RING finger复合物。我们发现秀丽隐杆线虫cullin cull -4需要将DNA复制限制在每个细胞周期一次。在缺乏CUL-4的情况下，细胞停留在S期并不断地重新复制其基因组DNA。cull -4限制基因组DNA复制的分子途径尚不清楚。相关cullin蛋白cul1和cul2在具有专用底物结合成分的多蛋白cullin/RING finger复合物中起作用。目前，在任何生物体中都没有描述过具有CUL-4功能的蛋白质复合物。秀丽隐杆线虫被用作研究CUL-4功能的模型系统，从而允许使用强大的遗传和分子方法。本实验将从三个方面阐明秀丽隐杆线虫CUL-4的分子功能。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、双杂交和抑制筛选将用于鉴定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学特征有望为调控DNA复制起始的基本过程提供重要的见解。最后，cull -4与真核DNA复制许可因子Cdc6和Cdt1的同源物之间的相互作用将被确定。除了对DNA复制的基本方面提供见解外，这项工作还有助于理解癌症的病因学，因为已发现人类CUL4A在乳腺癌中过度表达，而致癌基因的基因扩增（涉及DNA再复制）是多种癌症的一个促成因素。

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.