Regulator of DNA Replication & Gene Expression in Yeast

描述（由申请人提供）：DNA复制和基因表达的协调是细胞增殖调控的核心。协调这两个过程的策略是在两个过程中让相同的监管机构参与。这种双功能调节剂的典型例子是大肠杆菌dna和SV40大T抗原，它们同时具有复制起始和基因表达的调节剂的功能。Mcm1是一种MADS盒转录因子，调控细胞周期进程和DNA复制所需的基因。其活性受糖酵解通量、养分有效性和环境胁迫的影响。Mcm1还在复制起点结合特定的元件，促进DNA复制的开始。在本提案中，研究了Mcm1在原产地使用监管中的直接作用。Mcm1根据其在有限条件下的占用率调节原点使用的假设将在基因组尺度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Mcm1在选定复制起点的基因组宽位置，3)识别差异激活的早期复制染色体起点。预复制复合体（pre-RC）在Mcm1上的募集/激活的依赖性将通过染色质免疫沉淀实验进行研究。Mcm1和pre-RC组分之间的相互作用将通过电泳迁移迁移试验（EMSA）和dna足迹分析。Mcm1对局部DNA和染色质结构的影响将通过原子力显微镜、电子显微镜和核小体作图来可视化。在细胞增殖过程中协调DNA复制和基因表达的双功能调节因子包括E2F-RB和Myb-130。在酵母中模拟这一策略可能为细胞增殖因子和肿瘤抑制因子的机制作用提供见解。众所周知，DNA复制失调会产生不良影响，从不受控制的细胞增殖到细胞衰老。不协调的DNA复制也与染色体凝聚、内聚和断裂的缺陷有关，所有这些缺陷都对基因组的完整性造成可怕的后果。因此，了解DNA复制的调控是研究基因组不稳定性（包括三体和癌症）导致的所有遗传疾病病因学的核心。

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.