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物（ORC）在真核生物细胞周期进程中的作用。ORC是DMA复制的关键组成部分，但也在其他细胞功能中发挥作用。特别是，我们最近已经证明，复杂的是至关重要的参与胞质分裂。我们发现最小的ORC亚基Orc 6定位于细胞膜和分裂细胞的卵裂沟。Orc 6与Septin蛋白Peanut（Pnut）相互作用，并且除了其复制功能之外还参与胞质分裂。本论文的具体目标如下：1）Orc 6-Pnut相互作用的分子分析。Orc 6和Pnut蛋白的保守序列基序的功能将被定义，并将确定Pnut在Orc 6定位中的作用。2)确定对DNA结合和复制重要的Orc 6的基序和序列。将确定DNA结合和复制所必需的Orc 6的关键残基，并在体外和体内证实其重要性。3)在细胞周期期间Orc 6的细胞内定位的分析。将研究Orc 6定位在细胞周期的不同阶段的功能意义。4)Orc 6的体内靶向突变和功能丧失表型分析。将使用靶向诱变以及其他方法，使我们能够模拟突变体表型，以研究Orc 6在体内的功能。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，也允许深入了解复制控制的发育和组织特异性方面。对复制和胞质分裂起始中所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合理设计的癌症治疗方法，并可能提供再生因衰老，疾病或损伤而丢失的细胞的方法。

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.