NUCLEAR STRUCTURE AND METAZOAN ORIGINS OF REPLICATION

核结构和后生动物复制起源

• 批准号: 6019416
• 负责人: David M Gilbert
• 金额: $ 20.16万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019416
• 关键词: DNA replication origin; DNA topoisomerases; Xenopus; cell component structure /function; cell cycle; cell nucleus; dihydrofolate reductase; fluorescence microscopy; fluorescent in situ hybridization; gene expression; nucleoproteins; tissue /cell culture

DNA replication is central to the life cycle of every living organism. Although considerable progress has been made in our understanding of how this process is regulated in simple organisms, regulatory mechanisms in higher eukaryotes remain largely unknown. By introducing Chinese hamster ovary (CHO) cell nuclei into Xenopus egg extracts, the applicant has produced the first cell-free system that will initiate DNA replication preferentially at a physiologically utilized origin of replication downstream of the dihydrofolate reductase (DHFR) gene. Recognition of this origin requires some component of the CHO nucleus that is assembled at a discrete point during G1-phase (Origin Decision Point, ODP), after replication licensing and prior to restriction point control. After each mitosis, the cell must re-assemble a highly organized and functionally compartmentalized nucleus. In particular, replication takes place at fixed sites within the nucleus that consist of multiple coordinately regulated replicons joined to large (approximately 0.1 microns) multiprotein complexes. Our working hypothesis predicts that ODP represents the joining of replication origins to this multiprotein complex. To address this hypothesis, we will pulse label CHO cells with BrdU within the first 10 minutes of S- phase and chase these cells through to the following mitosis. Synchronized populations of CHO cells, containing BrdU-tagged origin- proximal sequences, will then be collected at various times during G1- phase and analyzed by fluorescence microscopy for the time at which these sequences 1) re-establish their early S-phase pattern of foci, 2) become attached to a fixed nuclear substratum, 3) become functionally recognizable as the sites at which to begin DNA synthesis when nuclei from these cells are introduced into Xenopus egg extracts, and 4) first co-localize with antibodies directed against essential replication initiation factors. Parallel experiments will monitor the spatial position and attachment to the matrix of specific origin-containing and non-origin probes that decorate the DHFR replicon using Fluorescence In Situ Hybridization to CHO cells synchronized in different stages of G1- phase. Having established the sequence of these events, we will disrupt the assembly of the nucleus through 1) the controlled overexpression of dominant negative nuclear lamina proteins that have been shown to interfere with the initiation of replication and 2) treatment of cells with inhibitors of Topoisomerase II, one of which has been shown to inhibit the selection of origins at the ODP. We will then determine which steps in the assembly of functional replication origins are interrupted by these disruptions in nuclear structure.

DNA复制是每个生物体生命周期的核心。 尽管我们在理解如何 这一过程在简单的生物体中受到调节， 高等真核生物仍然是未知的。 通过介绍中文 仓鼠卵巢（CHO）细胞核转化为非洲爪蟾卵提取物，申请人 产生了第一个无细胞系统， 复制优先在生理上利用的起点， 二氢叶酸还原酶（DHFR）基因的下游复制。 识别这种起源需要CHO细胞核的某些成分 在G1阶段（原产地决策）的离散点组装 点，ODP），在复制许可之后和限制点之前 控制 每次有丝分裂后，细胞必须重新组装一个高度 有组织的功能分区的核心。 特别是， 复制发生在细胞核内的固定位点， 多个协同调节的复制子连接到大的 （约0.1微米）多蛋白复合物。 我们的工作 假设预测ODP代表复制的加入 这个多蛋白复合物的起源。 为了解决这个问题，我们 将在S-10的前10分钟内用BrdU脉冲标记CHO细胞。 使这些细胞进行有丝分裂。 CHO细胞的同步群体，含有BrdU标记的来源- 然后将在G1期间的不同时间收集近端序列， 相，并通过荧光显微镜分析， 这些序列1）重新建立它们的早期S期病灶模式，2） 附着在固定的核基质上，3）在功能上变得 可识别为当细胞核分裂时开始DNA合成的位点 从这些细胞中引入爪蟾卵提取物，和4）首先 与针对基本复制的抗体共定位 启动因素 平行实验将监测空间 特定来源的基质的位置和附着， 使用荧光探针修饰DHFR复制子， 与在G1-G4的不同阶段同步化的CHO细胞的原位杂交 相位 在确定了这些事件的顺序之后，我们将破坏 细胞核的组装是通过1） 显性负核纤层蛋白已被证明， 干扰复制的起始和2）处理细胞 拓扑异构酶II抑制剂，其中一种已被证明 抑制ODP的起源选择。 然后我们将决定 在功能性复制起点的组装中， 被这些核结构的破坏所打断