The Role of origin Recognition Complex in the Cell Cycle

描述(由申请人提供)： 这项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，但也在其他单元功能中发挥作用。特别是，我们最近证明了该复合体与胞质分裂密切相关。我们发现，最小的ORC亚基ORC6定位于细胞膜和分裂细胞的裂沟。ORC6与Septin蛋白花生相互作用，除了其复制功能外，还参与细胞质分裂。将追求以下具体目标：1)ORC6-Pnut相互作用的分子分析。将确定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。将确定与DNA结合和复制所必需的ORC6关键残基，并在体外和体内确认它们的重要性。3)分析ORC6在细胞周期中的细胞内定位。我们将研究ORC6在细胞周期不同阶段定位的功能意义。4)ORC6体内靶向性突变及功能缺失表型分析。我们将使用定向突变以及其他方法来模拟突变表型来研究ORC6在体内的功能。与果蝇合作将对这些研究有利，因为它允许使用生化和遗传方法，还允许深入了解复制控制的发育和组织特定方面。对复制和胞质分裂的启动所涉及的分子事件的了解将为最终控制这些过程提供基础。这样的控制可能会导致癌症的合理设计疗法，并可能提供再生因衰老、疾病或伤害而失去的细胞的手段。

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.