Regulation of Replication Origin Usage in Saccharomyces cerevisiae

DMA复制和基因表达的协调是细胞增殖调节的核心。 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.科尔DNA A和SV 40大T抗原， 其起到复制起始和基因表达的调节剂的作用。M1是一个MADS 盒转录因子，调节细胞周期进程和DMA复制所需的基因。其 活性响应于糖酵解通量、营养可用性和环境压力。Mcm1还结合 在复制起点的特定元件，以促进启动DMA复制。在这一建议中，一个直接 Mcm1在原产地使用的调节作用进行了研究。Mcm1调节起源的假说 基于其在限制条件下的占有率的使用将在基因组规模上使用三个 不同的方法：1）彻底分离选择性地被克隆的自主复制序列， 繁殖，2）分析Mcm1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源。招募/激活的依赖性 将通过染色质免疫沉淀研究Mcm1上复制前复合物（pre-RC 实验Mcm1和预RC组分之间的相互作用将通过以下方法进行分析： 电泳迁移率变动分析（EMSA）和DNA酶!脚印Mcm1对局部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 核小体作图新出现的协调DMA复制的双功能调节器的例子， 在细胞增殖期间表达的基因包括E2F-RB和Myb-130。在酵母中模拟这种策略可能 提供深入了解细胞增殖因子和肿瘤抑制因子的机制作用。 已知错误调节的DNA复制具有不利影响， 增殖到细胞衰老。不协调的DNA复制也与基因缺陷有关。 染色体凝聚、凝聚和断裂，所有这些都对基因组产生可怕的后果 完整因此，了解DNA复制的调控是研究病因的核心 所有遗传疾病的根源在于基因组的不稳定性，包括三体和癌症。

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.