Role of ORCA in DNA replication

ORCA 在 DNA 复制中的作用

• 批准号: 8895987
• 负责人: Supriya G Prasanth
• 金额: $ 26.23万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8895987
• 关键词: 7q22.1; Acute leukemia; Address; Binding; Biochemical; Biochemistry; Biological; Biology; Biomedical Research; Burkitt Lymphoma; Cancer Biology; Cancer Diagnostics; Cancer Etiology; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Nucleus; Cell Survival; Cell division; Cells; Centromere; Centrosome; Chromatin; Chromosome Segregation; Chromosomes; Complex; DNA; DNA biosynthesis; DNA replication origin; Data; Daughter; Dysmyelopoietic Syndromes; Ensure; Esophageal Squamous Cell Carcinoma; G1 Phase; Geminin; Genes; Genetic Materials; Genome Stability; Goals; Heterochromatin; Human; Human Herpesvirus 4; Image; In Vitro; Laboratories; Leucine-Rich Repeat; Life; Link; Loss of Heterozygosity; Maintenance; Malignant Neoplasms; Mammalian Cell; Maps; Molecular; Molecular Biology; Molecular Weight; Pathway interactions; Play; Positioning Attribute; Procedures; Protein Complex Subunit; Proteins; Recruitment Activity; Regulation; Replication Initiation; Role; Site; Structure; Testing; Time; Ubiquitination; base; cellular imaging; combat; daughter cell; experience; genome integrity; in vivo; innovation; novel; origin recognition complex; segregation; telomere; therapeutic target; transmission process; ubiquitin-protein ligase

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.

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于细胞存活至关重要。起源识别复合物 (ORC) 是一种六亚基蛋白质复合物，可作为在 DNA 复制起点组装多蛋白预复制复合物的着陆平台。 ORC 和其他人类复制起始蛋白已被证明是与伯基特淋巴瘤相关的 Epstein Barr 病毒 (EBV) 复制所必需的。除了在 DNA 复制中发挥真正的作用外，ORC 蛋白也是异染色质组织、中心体拷贝数维持、准确的细胞周期进程和细胞分裂所必需的。我们发现了一种新的 ORC 相关蛋白 ORCA/LRWD1，它映射到人类细胞中的 7q22.1，该区域在食管鳞状细胞癌中经常扩增，或者在骨髓增生异常和急性白血病中显示出重排和杂合性丢失。 ORCA 与 ORC 共定位于端粒和着丝粒，这些结构对于维持基因组完整性至关重要。人类细胞中 ORCA 的消耗会导致 ORC 蛋白与染色质的结合减少，并随后导致细胞在 G1 期积累。然而，ORCA 在 preRC 组装和稳定 ORC 染色质中的作用仍有待阐明。我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 在复制启动中所起的作用，以及其细胞周期调节是否确保每个细胞周期复制“一次且仅一次”。基于初步数据，我们的假设是，ORCA-ORC 相互作用对于 ORC 到染色质的特异性负载以及 G1 期前 RC 组装和 G1 期后细胞中染色质组织至关重要。为了检验这一假设，我们提出以下具体目标：1）确定ORCA在preRC组装中的作用； 2) 确定ORCA如何稳定/招募ORC到染色质； 3) 明确ORCA细胞周期调控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颖组件。该提案将分子生物学、生物化学与最先进的活细胞成像相结合，具有创新性。我在分子生物学/生物化学和活细胞成像方面的专业知识对于这个项目来说是非常独特和重要的。包括迷你染色体维持蛋白在内的几种复制起始蛋白被广泛用于癌症诊断目的，查明 ORC 和 ORCA 在癌症中的功能以及它们如何调节基因组稳定性至关重要。因此，该提议不仅对于理解复制和细胞周期的基础知识具有非常重要的意义 进展是协调的，但对于癌症生物学领域也具有重要意义。