Role of ORCA in DNA replication

描述(由申请人提供)： 遗传物质的准确复制及其与子核的忠实分离对细胞生存至关重要。起源识别复合体(Origin Recognition Complex，ORC)是一个由六个亚基组成的蛋白质复合体，它是在DNA复制起始处组装多蛋白复制前复合体的着陆点。ORC和其他人类复制起始蛋白已被证明是复制与Burkitt淋巴瘤相关的Epstein Barr病毒(EBV)所必需的。除了在DNA复制中的真正作用外，ORC蛋白还是异染色质组织、中心体拷贝数维持、准确的细胞周期进展和细胞分裂所必需的。我们已经鉴定出一种新的ORC相关蛋白ORCA/LRWD1，它定位于人类细胞中的7q22.1，这是一个在食道鳞状细胞癌中经常被扩增的区域，或者在骨髓异常增生症和急性白血病中显示重排和杂合性丢失。虎鲸与兽人共定位于端粒和着丝粒，这是维持基因组完整性的关键结构。从人类细胞中去除ORCA会导致ORC蛋白与染色质的结合减少，从而使细胞积累在G1期。然而，ORCA在前RC组装和稳定ORC对染色质的作用仍有待阐明。我的实验室的长期目标是了解哺乳动物细胞中DNA复制和染色质组织是如何协调的。本提案的目的是确定ORCA在复制启动中所扮演的角色，以及它的细胞周期调节是否确保每个细胞周期发生一次复制。我们的假设是，基于初步数据，ORCA-ORC相互作用对于ORC特异性地装载到染色质、G1中的前RC组装和后G1细胞中的染色质组织至关重要。为了验证这一假说，我们提出了以下具体目标：1)确定ORCA在前RC组装中的作用；2)确定ORCA如何稳定/招募ORC到染色质；以及3)确定ORCA的细胞周期调节机制及其生物学意义。我们的方法是创新的，因为我们将描述复制启动机制的一个新组件。这一建议是创新的，它结合了分子生物学、生物化学和最先进的活细胞成像技术。我在分子生物学/生物化学和活细胞成像方面的专业知识是非常独特的，对这个项目至关重要。包括微小染色体维持蛋白在内的几种复制起始蛋白正被广泛用于癌症诊断，准确定位ORC和ORCA在癌症中的功能及其如何调节基因组稳定性将是至关重要的。因此，这一建议不仅对理解复制和细胞周期的基础知识具有重要意义 进展是协调的，但对癌症生物学领域也很重要。

DESCRIPTION (provided by applicant): Accurate duplication of genetic material and its faithful segregation to daughter nuclei is of paramount importance for cell survival. Origin Recognition Complex (ORC) is a six-subunit protein complex that serves as the landing pad for the assembly of a multiprotein pre-replicative complex at DNA replication origins. ORC and other human replication initiation proteins have been shown to be required for replication of Epstein Barr virus (EBV) that is associated with Burkitt's lymphoma. Other than their bonafide roles in DNA replication, ORC proteins are required for heterochromatin organization, centrosome copy number maintenance, accurate cell cycle progression and cell division. We have identified a novel ORC associated protein, ORCA/LRWD1 that maps to 7q22.1 in human cells, a region that is frequently amplified in esophageal squamous cell carcinoma or shows rearrangements and loss of heterozygosity in myelodysplasia and acute leukemia. ORCA colocalizes with ORCs to telomeres and centromeres, structures critical for maintenance of genome integrity. Depletion of ORCA from human cells results in reduced binding of ORC proteins to chromatin and subsequent accumulation of cells in G1 phase. However, the roles of ORCA in preRC assembly and in stabilizing ORC to chromatin remain to be elucidated. The long-term goal of my laboratory is to understand how DNA replication and chromatin organization are coordinated in mammalian cells. The objective of the present proposal is to determine the roles played by ORCA in replication initiation and if its cell cycle regulation ensures that replication occurs 'once-and-oly-once' per cell cycle. Our hypothesis, based on preliminary data, is that ORCA-ORC interaction is crucial for specific loading of ORC to chromatin and for preRC assembly in G1 and chromatin organization in post-G1 cells. To test this hypothesis, we propose the following specific aims: 1) Determine the role of ORCA in preRC assembly; 2) Determine how ORCA stabilizes/recruites ORC to chromatin; and 3) Identify the mechanism of cell cycle regulation of ORCA and its biological significance. Our approach is innovative because we will characterize a novel component of the replication initiation machinery. This proposal is innovative in its combination of molecular biology, biochemistry with state of the art live cell imaging. My expertise in both molecular biology/ biochemistry and live cell imaging is quite unique and essential to this project. Several replication initiation proteins including Mini chromosome maintenance proteins are being used widely for cancer diagnostic purposes and it would be critical to pinpoint the function of ORC and ORCA in cancer and how they regulate genome stability. This proposal is therefore highly significant not only in understanding the basics of how replication and cell cycle progression is coordinated but also significant for the field of cancer biology.