Regulator of DNA Replication & Gene Expression in Yeast

DNA复制的调节者

• 批准号: 7088159
• 负责人: BIK-KWOON TYE
• 金额: $ 28.96万
• 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/7088159
• 关键词: DNA replication origin; Saccharomyces cerevisiae; atomic force microscopy; cell proliferation; chromatin immunoprecipitation; electron microscopy; fungal genetics; gel mobility shift assay; gene expression; genetic mapping; genetic regulation; protein structure function; transcription factor

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细胞增殖的调节至关重要。协调这两个进程的一项战略是让同一监管机构参与这两个进程。这种双功能调节剂的经典例子是E. coliDnaA和SV 40大T抗原，它们同时起复制起始和基因表达调节剂的作用。Mcm 1是一种MADS盒转录因子，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、营养可用性和环境胁迫有反应。Mcm 1还在复制起点结合特定元件以促进DNA复制的起始。在这个建议中，Mcm 1在原产地使用的监管中的直接作用进行了研究。Mcm 1在限制条件下基于其占用调节起点使用的假设将在基因组规模上使用三种不同的方法进行研究：1）彻底分离选择性繁殖的自主复制序列，2）分析Mcm 1在选定复制起点的全基因组位置，3）鉴定差异激活的早期复制染色体起点。将通过染色质免疫沉淀实验研究复制前复合物（pre-RC）对Mcm 1的募集/激活的依赖性。将通过电泳迁移率变动试验（EMSA）和DNA酶1足迹法分析Mcm 1与前RC组分之间的相互作用。Mcm 1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作图来可视化。在细胞增殖期间协调DNA复制和基因表达的双功能调节剂的新兴实例包括E2 F-RB和Myb-130。在酵母中模拟这种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。已知错误调节的DNA复制具有从不受控制的细胞增殖到细胞衰老的不利影响。不协调的DNA复制也与染色体凝聚、凝聚和断裂的缺陷有关，所有这些都对基因组的完整性产生可怕的后果。因此，理解DNA复制的调节是研究所有遗传疾病的病因学的核心，这些遗传疾病植根于基因组不稳定性，包括三体和癌症。