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The Role of the DNA Unwinding Element Binding Protein, DUE-B, in DNA Replication

DNA 解旋元件结合蛋白 DUE-B 在 DNA 复制中的作用

基本信息

批准号：
7846744
负责人：
Michael LEFFAK
金额：
$ 29.78万
依托单位：
WRIGHT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7846744
关键词：
Abnormal Cell Address Affect Amino Acyl Transfer RNA Bacteria Behavior Binding Biochemical Biological Models Boron Cell Cycle Cell division Cells Chromatin Chromosome Breakage Co-Immunoprecipitations Complex Cyclin-Dependent Kinases DNA DNA Binding DNA Modification Process DNA biosynthesis DNA damage checkpoint DNA replication origin DNA-Binding Proteins Data Dependence Disease Elements Enzymes Evolution Flow Cytometry Fluorescence Resonance Energy Transfer Fractionation Genome Genomic Instability Hela Cells Hereditary Disease Homologous Gene Human Immunoblotting Immunofluorescence Immunologic In Vitro Inherited Label Laboratories Life Ligands Malignant Neoplasms Malignant neoplasm of ovary Mass Spectrum Analysis Modeling Monitor Mutation N-terminal Nuclear Personality Phase Phase Transition Phosphorylation Protein Binding Protein Dephosphorylation Protein Phosphatase 2A Regulatory Subunit PR53 Proteins Publishing RNA Processing Regulation Replication Initiation Replication Origin Role Site Structure Structure of thyroid parafollicular cell Techniques Testing Time Transfer RNA Two-Hybrid System Techniques Work Xenopus Yeasts analog c-myc Genes chromatin immunoprecipitation egg helicase in vivo insight mutant novel ovarian neoplasm overexpression protein complex sperm cell synthetic construct

项目摘要

The initiation of DNA replication is a critical control point of the cell cycle, since abnormal replication initiation leads to genome instability, cancer and other inherited diseases. Accurate control of initiation requires the ordered assembly of prereplication protein and preinitiation protein complexes at origins of replication. We have identified a novel DNA unwinding element binding protein, DUE-B, which binds specifically to the essential DUE of the human c-myc replication origin and other origins. DUE-B is necessary for normal entry into the DNA synthetic S phase of the cell cycle and for the initiation of replication. DUE-B functions after formation of the prereplication complex but before origin template unwinding, and is required to load the preinitiation complex proteins Cdc45 and TopBP1 at origins. DUE-B therefore shows strong similarity to the yeast Cdc45-loading protein Sld3, a key target of the S phase promoting cyclin- dependent kinases. We propose to test a model in which metazoan DUE-B is the functional homolog of Sld3. Adding significance to the role of DUE B in replication, we have found a strong correlation between DUE-B overexpression and ovarian cancer. Remarkably, DUE-B also shows a second personality; the structure of the N-terminal 75% of the protein has been strongly conserved during evolution, and displays aminoacyl-tRNA proofreading activities found in yeast, bacteria and archae. We will use HeLa cells and Xenopus egg extracts to address several mechanistic aspects of the model in which DUE-B regulates the binding of the helicase activator Cdc45 to form the replication preinitiation complex, and in which the DUE-B-dependent loading of Cdc45 is a target of the intra-S phase DNA damage checkpoint. Our major analytical techniques will be immunoblotting, chromatin immunoprecipitation, quantitative PCR, FRET, and immunofluorescence. In Aim 1 we will analyze the binding of DUE-B and preinitiation complex proteins to replication origins, and the effect of the intra-S phase DNA damage checkpoint on DUE-B function. Aim 2 will characterize the effects of targeted structural mutations in DUE-B on its binding to protein ligands and its activation of replication origins.

DNA复制的起始是细胞周期的关键控制点，因为复制起始异常会导致基因组不稳定、癌症和其他遗传性疾病疾病。精确控制启动需要预复制的有序组装复制起点处的蛋白质和预起始蛋白质复合物。我们已经确定了一个新型 DNA 解旋元件结合蛋白 DUE-B，可特异性结合人类 c-myc 复制起点和其他起点的重要 DUE。 DUE-B 是正常进入细胞周期 DNA 合成 S 期和复制的启动。 DUE-B 在复制前复合体形成后发挥作用但在原始模板展开之前，需要加载预启动复合体蛋白质 Cdc45 和 TopBP1 的起源。因此，DUE-B 与酵母 Cdc45 负载蛋白 Sld3，S 期促进细胞周期蛋白的关键靶标依赖性激酶。我们建议测试一个模型，其中后生动物 DUE-B 是 SLD3。我们发现 DUE B 在复制中的作用非常重要， DUE-B 过表达与卵巢癌的相关性。值得注意的是，DUE-B 还展现出第二人格； 75%的蛋白质的N端结构在进化过程中高度保守，并显示氨酰基-tRNA 校对在酵母、细菌和古细菌中发现的活性。我们将使用海拉细胞和非洲爪蟾卵提取物来解决几个机械问题 DUE-B 调节解旋酶激活剂结合的模型的各个方面 Cdc45 形成复制预起始复合体，其中 DUE-B 依赖性 Cdc45 的负载是 S 期 DNA 损伤检查点的目标。我们的专业分析技术包括免疫印迹、染色质免疫沉淀、定量 PCR、FRET 和免疫荧光。在目标 1 中，我们将分析绑定 DUE-B 和预起始复合蛋白对复制起点的影响，以及 DUE-B 功能上的 S 期 DNA 损伤检查点。目标 2 将描述 DUE-B 中靶向结构突变对其与蛋白质配体结合的影响及其复制起点的激活。