Analysis of the Human c-myc Gene Replication Origin

人类c-myc基因复制起点分析

• 批准号: 7226647
• 负责人: Michael LEFFAK
• 金额: $ 27.89万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7226647
• 关键词: ATP phosphohydrolase; Active Sites; Affect; Apoptosis; Base Pairing; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biological Models; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Cycle Regulation; Cell Line; Cell Proliferation; Cells; Chemotherapy-Oncologic Procedure; Chimeric Proteins; Chromatin Structure; Complex; DNA; DNA Damage; DNA Structure; DNA biosynthesis; DNA chemical synthesis; DNA repair protein; DNA-Protein Interaction; Data; Deoxyribonuclease I; Deoxyribonucleases; Digestion; Dimerization; Down-Regulation; Drug Delivery Systems; Elements; Epigenetic Process; FLP recombinase; Gel Chromatography; Genome; Genomic Instability; Genomics; Globin; Hela Cells; Histone Deacetylase; Human; Human Cell Line; Hybrids; In Vitro; Kidney Neoplasms; Lead; Length; MYB gene; Mass Spectrum Analysis; Measures; Mediating; Medical Surveillance; Methods; Methylation; Microsatellite Repeats; Modeling; Modification; Molecular Weight; Monitor; Multiprotein Complexes; Mutagenesis; Mutate; Mutation; Numbers; Ovarian; Pattern; Phosphoamino Acids; Phosphopeptides; Plasmids; Polymerase Chain Reaction; Post-Translational Protein Processing; Protein Binding; Protein Overexpression; Proteins; Proto-Oncogene Proteins c-myb; Recruitment Activity; Replication Initiation; Replication Origin; Replication-Associated Process; Role; Sequence Deletion; Site; Specific qualifier value; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; System; TOP1 gene; Testing; X-Ray Crystallography; Xenopus; Yeasts; base; c-myc Genes; chromatin immunoprecipitation; chromatin remodeling; egg; helicase; histone acetyltransferase; human TYRP1 protein; insight; mutant; replicator; research study; size; transcription factor; tumor

DESCRIPTION (provided by applicant): DNA replication is essential for cell proliferation, and is under strict surveillance since abnormal replication can lead to genomic instability, tumor formation or apoptosis. Drugs that target the process of replication are central to cancer chemotherapy, and insight into the control of replication may lead to new treatment strategies. The primary cell cycle control of replication occurs at replicators and replication origins, where the initiation of DNA synthesis depends on the assembly of multiprotein complexes. The human c-myc replicator will be used as a model system. We previously identified the c-myc replicator and demonstrated its activity at the endogenous c-myc locus, in transfected plasmids in human cells, in plasmids replicated in vitro, and at an ectopic site in the HeLa genome. Recently, we used the yeast FLP recombinase to target mutagenized c-myc replicator constructs to a specific genomic site and identify sequences essential for origin activity. Using one of these sequences as bait in a yeast one-hybrid assay we discovered the c-myc DNA unwinding element binding protein DUE-B. We will continue to use the FLP recombinase system to generate clonal human cell lines with targeted integration of c-myc replicator constructs. We will test several aspects of a model in which epigenetic factors specify replication initiation sites and the DUE-B protein is involved in the loading of DNA repair proteins at presumptive replication forks. Aim 1 will alter the structure of the c-myc replicator by methylation or substitution mutagenesis; Aim 2 will assess the relationship between protein binding and replicator activity. All replicator mutants will be analyzed at the same chromosomal acceptor site. Replicator activity and structure will be analyzed by quantitative PCR, DNase digestion, and chromatin immunoprecipitation. In Aim 3 we will characterize the DUE-B protein and its role in DNA replication using physical, biochemical and immunological methods.

描述（由申请人提供）： DNA复制对于细胞增殖至关重要，并且受到严格的监测，因为异常复制会导致基因组不稳定性，肿瘤形成或凋亡。针对复制过程的药物是癌症化学疗法的核心，对复制的控制可能会导致新的治疗策略。复制的主要细胞周期控制发生在复制器和复制起源，其中DNA合成的起始取决于多蛋白复合物的组装。 人类C-MYC复制器将用作模型系统。我们先前鉴定了C-MYC复制器，并证明了其在人类细胞中转染的质粒，体外复制的质粒和HELA基因组的异位位点中的质粒中转染的质粒中的活性。最近，我们使用酵母FLP重组酶将诱变的C-MYC复制构建体靶向特定的基因组位点，并确定原始活性必不可少的序列。使用这些序列之一作为酵母单杂交测定法中的诱饵，我们发现了c-myc DNA放松元素结合蛋白DA tue-b。 我们将继续使用FLP重组酶系统来生成具有C-MYC复制器构建体有针对性整合的克隆人细胞系。 我们将测试一个模型的几个方面，在该模型中，表观遗传因子指定复制起始位点和Do-B蛋白参与推定复制叉时DNA修复蛋白的负载。 AIM 1将通过甲基化或取代诱变改变C-MYC复制器的结构； AIM 2将评估蛋白质结合与复制剂活性之间的关系。所有复制子突变体将在同一染色体受体部位进行分析。复制剂的活性和结构将通过定量PCR，DNase消化和染色质免疫沉淀分析。在AIM 3中，我们将使用物理，生化和免疫学方法来表征Rue-B蛋白及其在DNA复制中的作用。