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; 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。对DNA复制的描述及其与地球上所有生物体中的细胞分裂的联系 基因组复制是一本针对高级本科生，研究生和专业科学家的全面且可读的教科书。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重复的基本原理。它包括概念，机制，进化和疾病。 2。关于确定DNA复制在哪里开始的原点识别复合物的研究 当六亚基原产识别复合物（ORC）与DNA结合时，真核基因组重复的起始开始。但是，这种机制发生在体内和各个亚基的作用似乎在生物体之间显着差异。先前的研究鉴定了核中的可溶性人兽（2-5）复合物，与染色质结合的兽人（1-5）复合物，以及一种与其他兽人亚基结合弱（如果有的话）的ORC6蛋白。在这里，我们表明稳定的兽人（1-6）复合物也可以从人类细胞提取物中纯化，并且ORC6和ORC1每个都包含一个单个核定位信号，这对于核定位是必不可少的，但对于ORC组装而言不是。对于ORC6功能所必需的ORC6核定位信号是通过其依赖细胞周期蛋白依赖性激酶共有位点的磷酸化促进的，并与没有与其他ORC亚基共纯化的核转运蛋白相关。这些和其他结果支持一个模型，其中ORC6，ORC1和ORC（2-5）独立运输到核，可以在其中组装到ORC（1-6）或单独运行。 3。开发针对诱导人类细胞重新复制的小分子的高吞吐量筛选测定 先前的研究表明，在不可能源自正常组织的细胞的情况下，可以在人类癌中衍生的细胞中诱导DNA再复制。由于DNA重新复制会诱导细胞死亡，因此可以将该策略应用于潜在的抗癌疗法。因此，开发了一种适合高通量筛选的成像测定法，该测定在完整细胞的核中测量超过四个基因组等效物的DNA复制并索引细胞增殖。通过筛选在正常和肿瘤衍生的细胞上的1,280个生物活性分子的文库来验证该测定法比检测过量DNA复制的当前方法更敏感。该屏幕鉴定出已知的过量DNA复制诱导剂，例如微管动力学的抑制剂，以及诱导正常和癌细胞中多余DNA复制的新型化合物。此外，确定了两种化合物，它们在癌细胞中选择性地诱导过量的DNA复制，一种在癌细胞中选择性诱导内猫。因此，该测定方法为发现化合物的发现提供了一种新方法，可用于研究基因组重复和治疗癌症的调节。 4。CHK1，响应DNA损伤的细胞周期出口的蛋白激酶也可以防止在没有DNA损伤的情况下细胞周期出口。 滋养细胞（TS）细胞在成纤维细胞生长因子-4的情况下增殖，但在不存在的情况下，它们分化为多倍体滋养细胞巨型（TG）细胞，这些细胞（TG）细胞仍然可行但无增殖。分化与CDK特异性抑制剂p21和p57的表达一致。其中p57对于从有丝分裂细胞循环转换为内鼠是必不可少的。在这里，我们表明，在没有诱导DNA损伤的情况下，检查点激酶1（CHK1），这是一种预防有丝分裂响应DNA损伤所必需的酶，可作为一种有丝分裂原依赖性蛋白激酶，可通过抑制P21和p27的表达，但不抑制P27，但CDK-MIT cyk cyk in Cyk in Cycy cyk in Cycy cys cys cys cys cy cys cys cys cys依赖有丝分裂原依赖性蛋白激酶。 CHK1在特定位点磷酸化P21和P57蛋白，从而将它们靶向26S蛋白酶体降解。 TG细胞缺乏CHK1，并且在TG细胞中恢复CHK1活性抑制了p57的表达并恢复有丝分裂。因此，CHK1是“ G2限制点”的一部分，该点可以防止编程用于末端分化的细胞中的过早细胞周期退出，这是CHK2无法扮演的角色。