Regulation of Replication Origin Usage in Saccharomyces cerevisiae

描述(申请人提供)：DNA复制和基因表达的协调是调节细胞增殖的中心。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。这种双重功能调节的经典例子是大肠杆菌DNAA和SV40大T抗原，它们既具有复制启动调节功能，又具有基因表达调节功能。Mcm1是一个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、营养物质的可利用性和环境胁迫做出反应。Mcm1还与复制起始处的特定元件结合，以促进DNA复制的启动。在这项提案中，研究了Mcm1在原产地使用调节中的直接作用。Mcm1根据其在限制条件下的占有率调节起始点使用的假设将在基因组水平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始点。通过染色质免疫沉淀实验研究复制前复合体(Pre-RC)对Mcm1的招募/激活的依赖性。Mcm1与前RC组分之间的相互作用将通过电泳迁移率改变分析(EMSA)和DNase1足迹进行分析。Mcm1对局部DNA和染色质结构的影响将通过原子力显微镜、电子显微镜以及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控因子的新例子包括E2F-Rb和Myb-130。在酵母中对这一策略进行建模可能会对细胞增殖因子和肿瘤抑制因子的机制作用提供深入的了解。众所周知，错误调控的DNA复制具有从不受控制的细胞增殖到细胞衰老的各种不利影响。不协调的DNA复制也与染色体凝聚、凝聚和碎裂的缺陷有关，所有这些都会对基因组完整性造成可怕的后果。因此，了解DNA复制的调控对于研究所有起源于基因组不稳定的遗传病的病因学是至关重要的，包括三体和癌症。

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.