Regulation of Replication Origin Usage in Saccharomyces cerevisiae

DNA复制和基因表达的协调是细胞增殖调控的核心。 协调这两个过程的战略是让相同的监管者参与这两个过程。 这种双功能调节子的经典例子是E.coll DNAA和SV40大T抗原， 它们既具有复制起始调节功能，又具有基因表达调节功能。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。它的 活性对糖酵解通量、营养可获得性和环境胁迫做出反应。Mcm1还结合 复制起始处的特定元素，以促进DMA复制的启动。在这项建议中，直接 研究了Mcm1在原产地使用调节中的作用。Mcm1调控起源的假说 基于其在限制条件下的占有率的使用将在基因组规模上进行调查，使用三个 不同的方法：1)彻底分离选择性地自主复制的序列 繁殖，2)分析Mcm1在选定复制起始处的全基因组位置，3)鉴定 差异激活的早期复制染色体起源。对招募/激活的依赖 Mcm1上的复制前复合体(Pre-RC)将通过染色质免疫沉淀进行研究 实验。将通过以下方式分析Mcm1与Pre-RC组件之间的相互作用 电泳迁移率改变分析(EMSA)和DNA酶！脚印。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 核小体图谱。新出现的双功能调节器，可协调DMA复制和 细胞增殖过程中的基因表达包括E2F-Rb和Myb-130。在酵母中对这种策略进行建模可能会 提供对细胞增殖因子和肿瘤抑制因子的机制作用的见解。 众所周知，错误调控的DNA复制会对不受控制的细胞产生不利影响 从增殖到细胞衰老。不协调的DNA复制也与基因缺陷有关 染色体凝聚、凝聚和碎裂，所有这些对基因组都有可怕的后果 正直。因此，了解DNA复制的调节对于病因学的研究至关重要 在所有根植于基因组不稳定的遗传病中，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.