人类c-myc基因复制起点分析-猫眼课题宝

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

项目类别：

财政年份：

1996

资助国家：

美国

项目状态：

已结题

起止时间：

1996-03-01 至 2009-04-30

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

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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.

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