NUCLEAR STRUCTURE AND METAZOAN ORIGINS OF REPLICATION

DNA复制是每一种生物生命周期的核心。 尽管在理解我们是如何 这一过程在简单的生物体中受到调节，调节机制在 高等真核生物在很大程度上仍是未知的。通过引入中文 仓鼠卵巢(CHO)细胞核制成非洲爪哇卵提取液，申请者 已经制造出了第一个无细胞系统，它将启动DNA 优先在物理上利用的起始处复制 二氢叶酸还原酶(DHFR)基因下游的复制。 识别这一起源需要CHO核的某些成分 在G1阶段期间在离散的点处组装(原点判定 点)、复制许可之后和限制点之前 控制力。在每次有丝分裂后，细胞必须重新组装一个高度 有组织的和功能上划分的核心。特别是， 复制在核内的固定位置进行，这些位置包括 多个协调调控的复制体连接到大的 (约0.1微米)多蛋白复合体。我们的工作 假说预测，ODP值代表复制的联接 起源于这种多蛋白复合体。为了解决这一假设，我们 将在S的头10分钟内用BrdU脉冲标记CHO细胞- 使这些细胞进入有丝分裂的阶段，直到随后的有丝分裂。 CHO细胞的同步群体，包含BrdU标记的起源- 近端序列，然后将在G1期间的不同时间收集- 并用荧光显微镜分析了它的时间 这些序列1)重建了其早期的S期病灶模式，2) 附着在固定的核基质上，3)在功能上 可识别为当细胞核时开始DNA合成的位置 从这些细胞中导入非洲爪哇卵提取液，4)首先 与针对基本复制的抗体共同定位 启蒙因素。平行实验将监测空间 定位和附着于特定原点的母体--包含和 使用荧光素修饰DHFR复制子的非起始探针 与CHO细胞的原位杂交在G1期不同时期同步进行 相位。在确定了这些事件的顺序后，我们将扰乱 通过1)受控的过表达来组装细胞核 显性负核层蛋白，已被证明 干扰复制的启动和2)细胞的治疗 拓扑异构酶II的抑制剂，其中一种已被证明 禁止在ODP处选择原点。然后我们将确定 组装功能复制源的步骤有哪些 被核结构的这些破坏所打断。

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.