Establishment of a Replication Timing Program in Mammalian Cells

这个项目的总体目标是阐明染色体DNA复制的计时程序是如何建立的。有人认为，在早期G1期序列的重新定位创建了微域，为复制的启动建立了阈值。从这个模型中得出的具体假设将得到检验。第一个项目将研究核内序列的重新定位是否与染色质的重塑有关。这项工作将确定早期和晚期复制染色质的差异DNaseI可及性是否在早期G1期建立。它还将检验组蛋白的低乙酰化或与染色质结构域蛋白HP1的结合是否是建立或维持异染色质的晚期复制时间所必需的。第二个项目将测试这一假设，即染色体结构通过设置阈值来限制起始蛋白对起始子集的访问，重点是哺乳动物的CDC45同源物和CDC7/Dbf4复合体。这些复制起始蛋白被认为是调控酿酒酵母S期进程中起始事件的关键靶点。这个项目将研究这些蛋白质的过度表达是否可以提前哺乳动物细胞中的复制计时程序。最后一个项目将使用蛋白质组学方法，结合体外复制计时分析，为建立复制计时程序的蛋白质确定新的候选蛋白质。工作模型预测，核内微环境是通过特定蛋白质与染色质的结合而产生的。这个项目将确定那些与染色质相关的蛋白质，在相对较短的结构域重新定位期间。它还将利用无细胞系统来丰富那些对建立复制计时程序至关重要的蛋白质。蛋白质组学提供了一种潜在的强大手段来解决复杂的生物学问题，这些问题不容易用传统的遗传和生化方法来解决。因此，这个项目也将考验这项新技术的力量。染色体的结构与它们在细胞中必须执行的功能有什么关系？染色体可以划分为独立的区域。当它们在一个称为DNA复制的过程中复制时，有一个定义的复制结构域的顺序。此外，通过从域中的多个站点同步开始复制，可以同时复制单个域中的所有DNA。有趣的是，在任何给定的细胞类型中没有使用的结构域最后被复制，而那些活跃的结构域被首先复制，这些结构域被复制的顺序在生物体发育的关键阶段发生变化。由于缺乏工具，在开发过程中很难研究结构域复制时间的变化与这些结构域功能之间的关系。最近，一种可以在试管中进行染色体复制的系统被开发出来，并被证明能够准确地概括染色体结构域的复制程序。这些研究为研究基因表达、染色体结构和DNA复制之间的关系开辟了一条令人兴奋的新途径。该项目将检验一种假设，即以前被认为调节染色体结构域结构的蛋白质可能直接调节这些结构域的复制程序。

The overall objective of this project is to elucidate how the timing program for the replication of chromosomal DNA is established. It is proposed that repositioning of sequences in early G1-phase creates microdomains that establish thresholds for the initiation of replication. Specific hypotheses derived from this model will be tested. The first project will be to examine whether the re-positioning of sequences in the nucleus is associated with a remodeling of chromatin. This work will determine whether the differential DNaseI accessibility of early- and late-replicating chromatin is established in early G1-phase. It will also examine whether hypoacetylation of histones or association with the chromo-domain protein HP1 is necessary to establish or maintain the late-replication time of heterochromatin. A second project will test the hypothesis that chromosome architecture mediates replication timing by setting thresholds that restrict the access of initiation proteins to a subset of origins, focusing on mammalian homologues of Cdc45 and the Cdc7/Dbf4 complex. These replication initiation proteins have been implicated as key targets for regulating initiation events during S-phase progression in S. cerevisiae. This project will examine whether over-expression of these proteins can advance the replication timing program in mammalian cells. The final project will use a proteomics approach, combined with an in vitro replication timing assay, to identify novel candidates for proteins that establish a replication timing program. The working model predicts that intranuclear microenvironments are created through the association of specific proteins with chromatin. This project will identify those proteins that associate with chromatin during the relatively brief period of domain re-positioning. It will also exploit a cell-free system to enrich for those proteins essential to the establishment of a replication timing program. Proteomics offers a potentially powerful means to approach complex biological problems that do not lend themselves easily to traditional genetic and biochemical approaches. Hence, this project will also test the power of this novel technology. How is the structure of chromosomes related to the functions that they have to carry out in the cell? Chromosomes can be divided into independent domains. When they duplicate in a process called DNA replication, there is a defined order in which the domains are replicated. Furthermore, all DNA within a single domain is replicated simultaneously, coordinated by starting replication synchronously from many sites within the domain. Intriguingly, domains that are not in use in any given cell-type are replicated last, while those that are active are replicated first and changes in the order in which these domains are replicated take place at key stages during the development of organisms. The relationship between changes in the replication timing of domains and the functions of those domains during development has been very difficult to study, due to the lack of tools. Recently, a system in which replication of chromosomes can be carried out in a test tube was developed and shown to accurately recapitulate the replication program of chromosome domains. These studies have opened up an exciting new avenue of research into the relationship between gene expression, chromosome structure and DNA replication. This project will test the hypothesis that proteins previously thought to regulate the structure of chromosomal domains may directly regulate the replication program of those domains.