The Role of origin Recognition Complex in the Cell Cycle

起源识别复合物在细胞周期中的作用

• 批准号: 7025674
• 负责人: IGOR N CHESNOKOV
• 金额: $ 24.94万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7025674
• 关键词: DNA binding protein; DNA replication; Drosophilidae; RNA interference; SDS polyacrylamide gel electrophoresis; actin binding protein; arthropod genetics; cell cycle; cell line; cell morphology; gene deletion mutation; point mutation; protein localization; protein protein interaction; protein structure function; site directed mutagenesis; yeast two hybrid system

DESCRIPTION (provided by applicant): The long term goal of this research program is to define the role of the Origin Recognition Complex (ORC) in cell cycle progression in eukaryotes. The ORC is a critical component for DMA replication, but also has a role in other cell functions. In particular, we have recently demonstrated that the complex is critically involved in cytokinesis. We found that the smallest ORC subunit, Orc6, is localized to the cell membranes and cleavage furrows of dividing cells. Orc6 interacts with the septin protein Peanut (Pnut) and is involved in cytokinesis in addition to its replication functions. The following specific aims will be pursued: 1) molecular analysis of Orc6-Pnut interaction. The function of conserved sequence motifs of Orc6 and Pnut proteins will be defined and the role of Pnut in Orc6 localization will be determined. 2) Determining the motifs and sequences of Orc6 that are important for DNA binding and replication. Critical residues of Orc6 essential for DNA binding and replication will be identified and their importance confirmed both in vitro and in vivo. 3) Analysis of the intracellular localization of Orc6 during the cell cycle. Functional significance of Orc6 localization during different stages of the cell cycle will be studied. 4) Targeted mutagenesis of Orc6 in vivo and analysis of the loss of function phenotype. Targeted mutagenesis will be used as well as other methods that allow us to mimic the mutant phenotype to study Orc6 functions in vivo. Working with Drosophila will be advantageous for these studies as it allows the use of both biochemical and genetic methods and also permits insights into the developmental as well as tissue specific aspects of replication control. An understanding of the molecular events involved in the initiation of replication and cytokinesis will provide a basis for ultimately controlling these processes. Such control might lead to rationally designed therapies for cancer and may provide the means for regenerating cells lost due to aging, disease or injury.

描述（由申请人提供）： 该研究计划的长期目标是定义原始识别复合物（ORC）在真核生物中细胞周期进程中的作用。兽人是DMA复制的关键组成部分，但在其他细胞功能中也起作用。特别是，我们最近证明了该复合物与细胞因子有关键有关。我们发现，最小的兽人亚基ORC6位于分裂细胞的细胞膜和裂解沟中。 ORC6与Septin蛋白花生（P Nut）相互作用，除了其复制功能外，还参与了细胞因子。将追求以下特定目的：1）ORC6-Pnut相互作用的分子分析。将定义ORC6和Pnut蛋白的保守序列基序的功能，并确定Pnut在ORC6定位中的作用。 2）确定对DNA结合和复制很重要的ORC6的基序和序列。将确定对DNA结合和复制必不可少的ORC6的关键残基，并且它们的重要性在体外和体内都证实。 3）分析细胞周期中ORC6的细胞内定位。将研究在细胞周期的不同阶段ORC6定位的功能显着性。 4）靶向ORC6体内的诱变和功能表型丧失的分析。将使用靶向诱变以及其他方法，使我们能够模仿突变表型在体内研究ORC6的功能。使用果蝇对这些研究将是有利的，因为它允许使用生化和遗传学方法，并且还允许对复制控制的发育和组织特定方面进行见解。对启动复制和细胞因子涉及的分子事件的理解将为最终控制这些过程提供基础。这种控制可能会导致为癌症进行合理设计的疗法，并可能为由于衰老，疾病或损伤而失去的细胞而提供手段。