Regulation of Replication Origin Usage in Saccharomyces cerevisiae

酿酒酵母复制起点使用的调控

• 批准号: 7596349
• 负责人: BIK-KWOON TYE
• 金额: $ 28.04万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7596349
• 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; DNA Binding; DNA biosynthesis; DNA-Directed DNA Polymerase; Defect; Deoxyribonucleases; Dependence; Electron Microscopy; Electrophoretic Mobility Shift Assay; Elements; 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; Nutrient; Pattern; Phase; Plant Roots; Play; Pre-Replication Complex; Process; Property; Proteins; 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; genome-wide; helicase; in vivo; insight; mutant; origin recognition complex; programs; research study; transcription factor

Coordination of DMA 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. coll 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 DMA 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 DMA 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 Dnase! footprinting. Influence of Mcm1 on the local DMA 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 DMA 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.

DMA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E. coll DNAA和SV40大T抗原， 这既服务于复制起始和基因表达的调节剂的功能。 MCM1是个疯子 盒转录因子调节细胞周期进程和DMA复制所需的基因。它是 活性对糖酵解通量，营养利用率和环境应力有反应。 MCM1也绑定 复制起源的特定元素，以促进DMA复制的启动。在此提案中，直接 研究了MCM1在起源使用法规中的作用。 MCM1调节起源的假设 根据限制条件下的占用率的用法将使用三个基因组量表进行研究 不同的方法：1）详尽隔离自主复制序列的序列 传播，2）分析在选定复制起源处MCM1的基因组宽位置，3） 差异激活的早期复制染色体起源。招募/激活的依赖性 MCM1上的恢复前复合物（PRE-RC）将通过染色质免疫沉淀研究 实验。 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法（EMSA）和DNase！足迹。 MCM1对本地DMA的影响 和染色质结构将通过原子力显微镜，电子显微镜以及 核小体映射。双重功能调节器的新兴示例，这些调节器协调DMA复制和 细胞增殖过程中的基因表达包括E2F-RB和MYB-130。在酵母中对此策略进行建模可能 提供有关细胞增殖因子和肿瘤抑制因子的机械作用的见解。 已知DNA复制错误的不良反应从不受控制的细胞不良反应 扩散到细胞衰老。不协调的DNA复制也已与缺陷有关 染色体凝结，内聚力和碎片化，所有这些都对基因组产生可怕的后果 正直。因此，了解DNA复制的调节对于研究的研究至关重要 在所有基因组不稳定性（包括三体和癌症）中的遗传疾病中。