Regulation of Replication Origin Usage in Saccharomyces cerevisiae

酿酒酵母复制起点使用的调控

基本信息

批准号：
7197986
负责人：
BIK-KWOON TYE
金额：
$ 28.06万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7197986
关键词：
Adverse effects Affinity Atomic Force Microscopy Binding Boxing Cell Aging Cell Cycle Progression Cell Proliferation Cell Proliferation Regulation Cell division Cells Chromatin Chromatin Structure Chromosome Condensation Chromosomes Complex Condition DNA DNA Binding DNA biosynthesis DNA chemical synthesis DNA-Directed DNA Polymerase Defect Dependence Electron Microscopy Electrophoretic Mobility Shift Assay Elements Escherichia coli Etiology Frequencies Gene Expression Genes Genome Genomic Instability Genomics Hereditary Disease In Vitro Laboratories Large T Antigen Length Link Location Maintenance Malignant Neoplasms Maps Mediating Modeling Molecular Nucleosomes Numbers Nutrient Pattern Phase Plant Roots Play Pre-Replication Complex Process Property Proteins Range Rate Recruitment Activity Regulation Replication Initiation Replication Initiation Gene Replication Origin Research Personnel Role Saccharomyces cerevisiae Simian virus 40 Site Stress Testing Trisomy Tumor Suppressor Proteins Yeasts autonomously replicating sequence base cell growth chromatin immunoprecipitation cohesion helicase in vivo insight mutant origin recognition complex programs research study transcription factor

项目摘要

DESCRIPTION (provided by applicant): Coordination of DNA replication and gene expression is central to the regulation of cell proliferation. A strategy for the coordination of these two processes is to engage the same regulators in both processes. Classical examples of such dual functional regulators are the E. coli DnaA and the SV40 large T antigen, which serve both the functions of regulators of replication initiation and gene expression. Mcm1 is a MADS box transcription factor which regulates genes required for cell cycle progression and DNA replication. Its activity is responsive to glycolytic flux, nutrient availability and environmental stresses. Mcm1 also binds specific elements at replication origins to promote initiation of DNA replication. In this proposal, a direct role for Mcm1 in the regulation of origin usage is investigated. The hypothesis that Mcm1 regulates origin usage based on its occupancy under limiting conditions will be investigated at a genomic scale using three different approaches: 1) to exhaustively isolate autonomously replicating sequences that are selectively propagated, 2) to analyze the genome wide locations of Mcm1 at selected replication origins, 3) to identify differentially activated early replicating chromosomal origins. Dependence of the recruitment/activation of the pre-replication complex (pre-RC) on Mcm1 will be investigated by chromatin immunoprecipitation experiments. Interactions between Mcm1 and components of the pre-RC will be analyzed by electrophoretic mobility shift assay (EMSA) and Dnase1 footprinting. Influence of Mcm1 on the local DNA and chromatin structures will be visualized by atomic force microscopy, electron microscopy as well as nucleosome mapping. Emerging examples of dual functional regulators that coordinate DNA replication and gene expression during cell proliferation include E2F-RB and Myb-130. Modeling this strategy in yeast may provide insights into the mechanistic actions of cell proliferation factors and tumor suppressors. Mis-regulated DNA replication is known to have adverse effects ranging from uncontrolled cell proliferation to cellular senescence. Uncoordinated DNA replication has also been linked to defects in chromosome condensation, cohesion and fragmentation, all of which have dire consequences on genome integrity. Therefore, understanding the regulation of DNA replication is central to the study of the etiology of all genetic diseases rooted in genome instability including trisomy and cancer.

描述（由申请人提供）：DNA复制和基因表达的协调对于调节细胞增殖至关重要。协调这两个过程的一种策略是在这两个过程中参与相同的调节器。这种双重功能调节剂的经典示例是大肠杆菌DNAA和SV40大T抗原，它们既服务于复制起始的调节剂和基因表达的调节剂。 MCM1是一种MADS盒转录因子，它调节细胞周期进程和DNA复制所需的基因。它的活性对糖酵解通量，养分可用性和环境应力有反应。 MCM1还结合了复制起源时的特定元素，以促进DNA复制的启动。在此提案中，研究了MCM1在原产地使用中的直接作用。将使用三种不同的方法以基因组量表研究MCM1根据其占用条件调节其起源的假设：1）详尽地分离自动复制序列的序列，有选择性地传播，2）分析MCM1基因组宽位置的序列选定的复制起源，3）确定差异激活的早期复制染色体起源。染色质免疫沉淀实验将研究恢复复合物（PRE-RC）对MCM1的募集/激活的依赖性。 MCM1和PER-RC组件之间的相互作用将通过电泳移动性转移测定（EMSA）和DNASE1足迹进行分析。 MCM1对局部DNA和染色质结构的影响将通过原子力显微镜，电子显微镜以及核小体映射来观察。在细胞增殖过程中协调DNA复制和基因表达的双重功能调节剂的新兴实例包括E2F-RB和MYB-130。在酵母中对此策略进行建模可能会提供有关细胞增殖因子和肿瘤抑制剂的机械作用的见解。已知DNA复制错误的不良反应范围从不受控制的细胞增殖到细胞衰老。不协调的DNA复制也与染色体凝结，内聚力和碎片化的缺陷有关，所有这些都对基因组完整性产生了可怕的后果。因此，了解DNA复制的调节对于研究了植根于基因组不稳定性（包括三体疾病和癌症）的所有遗传疾病的病因的研究至关重要。