Initiation Of DNA Replication In Mammalian Chromosomes

哺乳动物染色体中 DNA 复制的起始

• 批准号: 8351104
• 负责人: Melvin DePamphilis
• 金额: $ 84.75万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8351104
• 关键词: 26S proteasome; Adult; Age; Animals; Apoptosis; Bacteria; Binding; Binding Sites; Biological Assay; Blood Platelets; CDKN1C gene; CHEK2 gene; Carrier Proteins; Cell Cycle; Cell Death; Cell Extracts; Cell Nucleus; Cell Proliferation; Cell division; Cells; Checkpoint kinase 1; Chromatin; Chromatin Structure; Chromosomes; Cleaved cell; Complex; Consensus; Cyclin-Dependent Kinases; DNA; DNA Damage; DNA Methylation; DNA Repair Gene; DNA Sequence; DNA Virus Infections; DNA biosynthesis; DNA damage checkpoint; DNA replication origin; Deoxyribonucleotides; Development; Disease; Embryo; Enzymes; Epigenetic Process; Event; Evolution; Fishes; Generations; Genes; Genetic; Genetic Transcription; Genome; Genomic Instability; Genomics; Giant Cells; Goals; Human; Image; Individual; Inherited; Laboratories; Libraries; Life; Location; Malignant Neoplasms; Mammalian Chromosomes; Measures; Megakaryoblast; Megakaryocytes; Methods; Microtubules; Mitogens; Mitosis; Mitotic Cell Cycle; Modeling; Molecular; Mutation; Normal Cell; Normal tissue morphology; Nuclear; Nuclear Localization Signal; Organism; Phosphorylation; Planet Earth; Play; Polyploidy; Proliferating; Protein Binding; Protein Kinase; Proteins; Rana; Regulation; Regulatory Pathway; Replication Origin; Replication-Associated Process; Reporting; Role; Scientist; Screening procedure; Site; Staging; Stem cells; Structure; Textbooks; Time; Tumor-Derived; accomplished suicide; anti-cancer therapeutic; cancer cell; cancer therapy; cell type; cyclin-dependent kinase inhibitor 1B; density; fly; graduate student; high throughput screening; human ORC1L protein; human disease; in vivo; indexing; inhibitor/antagonist; initiation site of DNA replication; natural Blastocyst Implantation; novel; novel strategies; nucleocytoplasmic transport; origin recognition complex; premature; prevent; progenitor; programs; protein aggregate; proto-oncogene protein kfgf; response; small molecule; transcription factor; trophoblast

1. A description of DNA replication and its linkage to cell division in all living organisms on planet Earth Genome Duplication is a comprehensive and readable textbook for advanced undergraduates, graduate students and professional scientists. It describes the underlying principles governing genome duplication from the simplest single cell bacterium to the most complex multicellular organism. It includes concepts, mechanisms, evolution and disease. 2. Studies on the Origin Recognition Complex that determines where DNA replication begins Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2-5) complex in the nucleus, an ORC(1-5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1-6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2-5) are transported independently to the nucleus where they can either assemble into ORC(1-6) or function individually. 3. Development of a high throughput screening assay for small molecules that induce DNA re-replication in human cells Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1,280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication, such as inhibitors of microtubule dynamics, and novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer. 4. CHK1, the protein kinase that induces cell cycle exit in response to DNA damage can also prevent cell cycle exit in the absence of DNA damage. Trophoblast stem (TS) cells proliferate in the presence of fibroblast growth factor-4, but in its absence, they differentiate into polyploid trophoblast giant (TG) cells that remain viable but nonproliferative. Differentiation is coincident with expression of the CDK-specific inhibitors p21 and p57; of which p57 is essential for switching from mitotic cell cycles to endocycles. Here we show that, in the absence of induced DNA damage, checkpoint kinase-1 (CHK1), an enzyme essential for preventing mitosis in response to DNA damage, functions as a mitogen-dependent protein kinase that prevents premature differentiation of TS cells into TG cells by suppressing expression of p21 and p57, but not p27, the CDK-inhibitor that regulates mitotic cell cycles. CHK1 phosphorylates p21 and p57 proteins at specific sites, thereby targeting them for degradation by the 26S proteasome. TG cells lack CHK1, and restoring CHK1 activity in TG cells suppresses expression of p57 and restores mitosis. Thus, CHK1 is part of a 'G2 restriction point' that prevents premature cell cycle exit in cells programmed for terminal differentiation, a role that CHK2 cannot play.

1. 地球上所有生物体中 DNA 复制及其与细胞分裂的联系的描述 基因组复制是一本面向高年级本科生、研究生和专业科学家的综合性、可读性教科书。它描述了从最简单的单细胞细菌到最复杂的多细胞生物的基因组复制的基本原理。它包括概念、机制、进化和疾病。 2. 决定DNA复制开始位置的起源识别复合体的研究 当六亚基起源识别复合物 (ORC) 与 DNA 结合时，真核基因组复制就开始启动。然而，这种情况在体内发生的机制以及各个亚基所发挥的作用似乎在生物体之间存在显着差异。先前的研究鉴定了细胞核中的可溶性人类 ORC(2-5) 复合物、与染色质结合的 ORC(1-5) 复合物以及与其他 ORC 亚基结合较弱（如果有的话）的 Orc6 蛋白。在这里，我们证明稳定的 ORC(1-6) 复合物也可以从人类细胞提取物中纯化，并且 Orc6 和 Orc1 各自包含一个核定位信号，该信号对于核定位至关重要，但对于 ORC 组装则不然。 Orc6 核定位信号对于 Orc6 功能至关重要，通过其细胞周期蛋白依赖性激酶共有位点的磷酸化以及与 Kpna6/1（不与其他 ORC 亚基共纯化的核转运蛋白）的关联来促进。这些和其他结果支持一个模型，其中 Orc6、Orc1 和 ORC(2-5) 独立运输到细胞核，在那里它们可以组装成 ORC(1-6) 或单独发挥作用。 3. 开发诱导人类细胞 DNA 再复制的小分子高通量筛选方法 先前的研究表明，在正常组织细胞无法诱导的条件下，人类癌症细胞可以诱导 DNA 重新复制。由于 DNA 重新复制会诱导细胞死亡，因此该策略可用于发现潜在的抗癌疗法。因此，开发了一种适合高通量筛选的成像测定法，用于测量完整细胞核中超过四个基因组当量的 DNA 复制并指示细胞增殖。该检测方法通过在正常细胞和肿瘤来源的细胞上筛选包含 1,280 个生物活性分子的文库而得到验证，事实证明该检测方法比目前检测过量 DNA 复制的方法更灵敏。该筛选鉴定了已知的过度 DNA 复制诱导剂，例如微管动力学抑制剂，以及在正常细胞和癌细胞中诱导过度 DNA 复制的新型化合物。此外，还鉴定出两种化合物可在癌细胞中选择性诱导过量 DNA 复制，另一种化合物可在癌细胞中选择性诱导内循环。因此，该测定提供了一种发现可用于研究基因组复制调节和治疗癌症的化合物的新方法。 4. CHK1是一种蛋白激酶，它响应DNA损伤而诱导细胞周期退出，也可以在没有DNA损伤的情况下阻止细胞周期退出。 滋养层干 (TS) 细胞在成纤维细胞生长因子 4 存在的情况下增殖，但在缺乏成纤维细胞生长因子 4 的情况下，它们会分化为多倍体滋养层巨细胞 (TG)，该细胞仍能存活但不增殖。分化与 CDK 特异性抑制剂 p21 和 p57 的表达一致；其中p57对于有丝分裂细胞周期向内周期的转换至关重要。在这里，我们发现，在没有诱导 DNA 损伤的情况下，检查点激酶 1 (CHK1)（一种防止 DNA 损伤导致有丝分裂所必需的酶）可作为有丝分裂原依赖性蛋白激酶，防止 TS 细胞过早分化为 TG通过抑制 p21 和 p57 的表达来抑制细胞的表达，但不抑制 p27（调节有丝分裂细胞周期的 CDK 抑制剂）。 CHK1 在特定位点磷酸化 p21 和 p57 蛋白，从而靶向它们被 26S 蛋白酶体降解。 TG细胞缺乏CHK1，恢复TG细胞中的CHK1活性会抑制p57的表达并恢复有丝分裂。因此，CHK1 是“G2 限制点”的一部分，可防止细胞周期过早退出被编程为终末分化的细胞，而 CHK2 无法发挥这一作用。