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复制的启动是细胞周期的关键控制点，因为异常复制引发导致基因组不稳定性，癌症和其他遗传疾病。准确控制启动需要有序的预定装置复制起源的蛋白质和前启动蛋白复合物。我们已经确定了新型的DNA放松元素结合蛋白Due-B，该蛋白与B，该蛋白特异性结合至关重要的是人类C-MYC复制起源和其他起源。 Due-B是正常进入细胞周期的DNA合成阶段所必需的启动复制。形成预定复合物后的应有B功能但是在原点模板放松之前，需要加载前启动复合物蛋白质CDC45和TOPBP1在起源。因此，应得的B显示与酵母Cdc45载蛋白SLD3，S相促进细胞周期蛋白的关键靶标依赖激酶。我们建议测试一个模型，其中后生tudue-b是 SLD3。增加了应务B在复制中的作用的重要性，我们发现了一个强大的应有的B过表达与卵巢癌之间的相关性。值得注意的是，Due-B 还显示了第二个个性； N末端75％的蛋白质的结构具有在进化过程中得到了强烈的保守，并显示氨基酰基-TRNA校对在酵母，细菌和考古中发现的活动。我们将使用HeLa细胞和爪蟾鸡蛋提取物来解决几种机械该模型的各个方面，在该方面调节解旋酶激活剂的结合 cdc45形成复制前启动复合复合物，其中应依赖性依赖性 Cdc45的加载是Intra-S相DNA损伤检查点的目标。我们的专业分析技术将是免疫印迹，染色质免疫沉淀，定量PCR，FRET和免疫荧光。在AIM 1中，我们将分析绑定重复起源的育儿和预原始复合蛋白的作用以及 s Intra-S相DNA损伤检查点在Rue-B功能上。 AIM 2将表征靶向B中靶向结构突变对其与蛋白质配体的结合及其结合的影响复制起源的激活。