Regulation of Replication Origin Usage in Saccharomyces cerevisiae

酿酒酵母复制起点使用的调控

• 批准号: 7391551
• 负责人: BIK-KWOON TYE
• 金额: $ 28.07万
• 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 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7391551
• 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 Binding; DNA biosynthesis; DNA chemical synthesis; DNA-Directed DNA Polymerase; Defect; Deoxyribonuclease I; 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; 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

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细胞增殖的调节至关重要。 协调这两个流程的策略是让相同的监管机构参与这两个流程。 这种双功能调节剂的经典例子是大肠杆菌 DnaA 和 SV40 大 T 抗原， 它们具有复制起始和基因表达调节因子的功能。 Mcm1 是 MADS 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、营养可用性和环境压力有反应。 Mcm1 也结合 复制起点处的特定元件可促进 DMA 复制的启动。在这个提案中，直接 研究了 Mcm1 在来源使用调节中的作用。 Mcm1 调节起源的假设 基于其在限制条件下的占用的使用将使用三个在基因组规模上进行研究 不同的方法：1）彻底分离有选择性的自主复制序列 传播，2) 分析 Mcm1 在选定复制起点的全基因组位置，3) 识别 差异激活的早期复制染色体起源。招募/激活的依赖性 Mcm1 上的复制前复合物 (pre-RC) 将通过染色质免疫沉淀进行研究 实验。 Mcm1 和预 RC 组件之间的相互作用将通过以下方式进行分析 电泳迁移率变动分析 (EMSA) 和 DNA酶！足迹。 Mcm1对本地DMA的影响 染色质结构将通过原子力显微镜、电子显微镜以及 核小体作图。协调 DMA 复制和功能的双功能调节器的新兴示例 细胞增殖过程中表达的基因包括E2F-RB和Myb-130。在酵母中模拟这一策略可能会 提供对细胞增殖因子和肿瘤抑制因子的机制作用的见解。 已知 DNA 复制错误调控会产生一系列不利影响，包括不受控制的细胞 增殖导致细胞衰老。不协调的 DNA 复制也与以下缺陷有关： 染色体凝聚、凝聚和断裂，所有这些都会对基因组产生可怕的后果 正直。因此，了解DNA复制的调控对于病因学研究至关重要。 根源于基因组不稳定的所有遗传疾病，包括三体性和癌症。