Origin Recognition Complex in the Cell Cycle

细胞周期中的起源识别复合物

• 批准号: 6924109
• 负责人: IGOR N CHESNOKOV
• 金额: $ 25.43万
• 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/6924109
• 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确定起源识别复合体 (ORC) 在真核生物细胞周期进程中的作用。 ORC 是 DMA 复制的关键组件，但也在其他细胞功能中发挥作用。特别是，我们最近证明该复合物与胞质分裂密切相关。我们发现最小的 ORC 亚基 Orc6 位于细胞膜和分裂细胞的卵裂沟中。 Orc6 与 septin 蛋白 Peanut (Pnut) 相互作用，除了其复制功能外，还参与胞质分裂。我们将追求以下具体目标：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结合和复制重要的Orc6基序和序列。对 DNA 结合和复制至关重要的 Orc6 关键残基将被鉴定，并在体外和体内证实其重要性。 3)细胞周期中Orc6的细胞内定位分析。将研究 Orc6 定位在细胞周期不同阶段的功能意义。 4）Orc6体内定点突变及功能丧失表型分析。我们将使用定向诱变以及其他方法来模拟突变表型来研究 Orc6 的体内功能。与果蝇合作将有利于这些研究，因为它允许使用生化和遗传方法，并且还允许深入了解复制控制的发育和组织特异性方面。了解复制起始和胞质分裂所涉及的分子事件将为最终控制这些过程提供基础。这种控制可能会带来合理设计的癌症疗法，并可能提供再生因衰老、疾病或损伤而损失的细胞的方法。