Establishment of a Replication Timing Program in Mammalian Cells

哺乳动物细胞复制计时程序的建立

• 批准号: 0077507
• 负责人: David Gilbert
• 金额: $ 40万
• 依托单位: SUNY, Upstate Medical University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0077507&HistoricalAwards=false
• 关键词: Establishment; Replication; Timing; Program; Mammalian

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.

本项目的总体目标是阐明染色体DNA复制的时间程序是如何建立的。有人提出，重新定位的序列在早期G1期创建微域，建立复制启动的阈值。 将检验从该模型得出的具体假设。第一个项目将是检查细胞核中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。 它还将检查是否hypoacetylation的组蛋白或协会与色域蛋白HP 1是必要的，以建立或维持后期复制时间的异染色质。第二个项目将测试的假设，染色体结构介导的复制时间，通过设置阈值，限制访问起始蛋白质的一个子集的起源，重点是哺乳动物同源的Cdc 45和Cdc 7/Dbf 4复合物。 这些复制起始蛋白被认为是调节S期进展过程中起始事件的关键靶点。啤酒。该项目将研究这些蛋白质的过度表达是否可以促进哺乳动物细胞中的复制定时程序。最后的项目将使用蛋白质组学的方法，结合体外复制定时测定，以确定新的候选蛋白质，建立一个复制定时程序。 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蛋白质与染色质在相对较短的时期内结构域重新定位。 它还将利用无细胞系统来富集那些对建立复制定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，这些问题不容易通过传统的遗传和生物化学方法来解决。因此，该项目也将测试这项新技术的威力。 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？ 染色体可分为独立的结构域。 当它们在一个称为DNA复制的过程中复制时，这些结构域的复制有一个确定的顺序。 此外，单个结构域内的所有DNA同时复制，通过从结构域内的许多位点同步开始复制来协调。 有趣的是，在任何给定的细胞类型中不使用的结构域最后复制，而那些活跃的结构域首先复制，并且这些结构域复制顺序的变化发生在生物体发育的关键阶段。 由于缺乏工具，域复制时间的变化与开发过程中这些域的功能之间的关系一直很难研究。 最近，一个系统，其中染色体的复制可以在试管中进行的开发和显示，准确地概括了染色体域的复制程序。 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。 这个项目将测试一个假设，即以前认为调节染色体结构域的蛋白质可能直接调节这些结构域的复制程序。