Regulation of Replication Origin Usage in Saccharomyces cerevisiae

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。协调这两个流程的策略是让相同的监管机构参与这两个流程。这种双功能调节因子的经典例子是大肠杆菌 DnaA 和 SV40 大 T 抗原，它们同时具有复制起始和基因表达调节因子的功能。 Mcm1 是一种 MADS 盒转录因子，可调节细胞周期进程和 DNA 复制所需的基因。其活性对糖酵解通量、营养可用性和环境压力有反应。 Mcm1 还在复制起点结合特定元件以促进 DNA 复制的启动。在此提案中，研究了 Mcm1 在原产地使用监管中的直接作用。 Mcm1 根据其在限制条件下的占用情况调节起点使用的假设将使用三种不同的方法在基因组规模上进行研究：1）彻底分离选择性繁殖的自主复制序列，2）分析选定复制起点处 Mcm1 的全基因组位置，3）识别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) 的招募/激活对 Mcm1 的依赖性。 Mcm1 和 pre-RC 组件之间的相互作用将通过电泳迁移率变化测定 (EMSA) 和 Dnase1 足迹分析进行分析。 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节因子的新兴例子包括 E2F-RB 和 Myb-130。在酵母中模拟这种策略可以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，DNA 复制失调会产生一系列不利影响，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、凝聚和断裂的缺陷有关，所有这些都会对基因组完整性产生可怕的后果。因此，了解 DNA 复制的调控对于研究所有源于基因组不稳定的遗传疾病（包括三体性和癌症）的病因学至关重要。

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.