DNA REPLICATION, CELL CYCLE CONTROL AND GENOME FLUIDITY

DNA 复制、细胞周期控制和基因组流动性

• 批准号: 3309718
• 负责人: Geoffrey Myles Wahl
• 金额: $ 23.75万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3309718
• 关键词: DNA replication; DNA replication origin; binding proteins; cell cycle; chromosome deletion; chromosomes; cytogenetics; gene mutation; genetic regulatory element; hamsters; molecular cloning; natural gene amplification; nucleic acid sequence; polymerase chain reaction; recombinant DNA; restriction mapping; site directed mutagenesis; southern blotting; synchronous cell division; transfection

The long term objectives of this proposal is to understand how the initiation step of DNA replication is regulated. Previous studies investigated the molecular mechanisms underlying gene amplification, a type chromosomal rearrangement that does not occur in normal somatic cells, but occurs in many clinically important human cancers. A "bubble- breakage" model fore gene amplification evolved from these studies; it postulates that entry into 5-phase under metabolically limiting conditions precipitates the formation of amplicons by chromosome breakage within slowed or stalled replication bubbles. The amplicons are proposed to be autonomously replicating large chromosome fragments or small fragments such as double minute chromosomes (DMs) or their episomal precursors. This work further emphasized the need to identify the regions within which DNA replication initiates, and to develop functional assays to enable their molecular dissection. The first two Specific Aims propose to identify, isolate, and characterize the cis acting sequences that enable bidirectional DNA replication to initiate. Three putative initiation regions were identified and cloned from episomes during the past grant period. They do not enable autonomous replication of transfected sequences, but they do function in the genome. This raises the possibility that origin function might depend on an "imprinting" process occurring within a chromosome. A biochemical assay for origin activity, and a molecular strategy employing FLP, a yeast site specific recombinase adapted to function in mammalian cells, will be used to test this idea. Furthermore, one initiation region appears to contain a replication fork barrier which will be analyzed using biochemical and genetic strategies. The third specific aim proposes to investigate structure-function relationships in replication origins in their native chromosomal location. This is an essential goal to achieve, since one must ultimately have a means of investigating how sequences in the vicinity of the origin impact on origin activity. The strategy employs homologous recombination to produce specific alterations in regions shown in the first two specific aims to be important for origin function. These studies will be performed in cell lines that have been made hemizygous for the candidate origin region using one of several new methods for deleting specific regions of the mammalian genome. One method involves a combination of homologous and FLP-mediated recombination to generate targeted deletions of large genomic regions and to produce an autonomous episome harboring specific origins as a reciprocal The multifaceted approach to the analysis of replication origins within and outside of the chromosome should elucidate the minimal regions required for origin function, and enable a mob precise molecular understanding of those features that contribute to origin function and the initiation of DNA replication.

这项提案的长期目标是理解 DNA复制的起始步骤是受调控的。以前的研究 研究了基因扩增的分子机制，一个 在正常体细胞中不发生的类型染色体重排 细胞，但存在于许多临床上重要的人类癌症中。一个“泡沫-- 基因扩增的“断裂”模式是从这些研究发展而来的；它 假设进入新陈代谢受限的5个阶段 条件通过染色体断裂加速了扩增产物的形成 在缓慢或停滞的复制泡沫中。提出了扩增子的概念。 自主复制大的或小的染色体片段 片段，如双小染色体(DM)或它们的上体 先驱物。这项工作进一步强调了确定区域的必要性 DNA复制在其中启动，并开发功能分析 以使它们能够进行分子解剖。 前两个具体目标建议识别、隔离和 表征实现双向DNA的顺式作用序列 启动复制。三个可能的起始区是 在过去的资助期内，从附体中鉴定和克隆。他们 不支持转基因序列的自主复制，但它们 确实在基因组中发挥作用。这增加了起源的可能性 函数可能依赖于在 染色体。一种来源活性的生化分析，以及一种分子 使用FLP的策略，FLP是一种酵母位点特异性重组酶，适用于 在哺乳动物细胞中的功能，将被用于测试这一想法。此外， 一个起始区似乎包含复制叉状屏障，该复制叉状屏障 将使用生化和遗传策略进行分析。 第三个具体目的是提出研究结构-功能 复制起源与其天然染色体之间的关系 地点。这是一个必须实现的目标，因为一个人最终必须 有办法研究原点附近的序列是如何 对起源活动的影响。该策略采用同源重组 在前两个区域中产生特定的更改 特定的目标对于起源功能是重要的。这些研究将是 在已经使候选人半合子的细胞系中进行 使用几种新方法之一删除特定区域的原点区域 哺乳动物基因组的区域。一种方法涉及以下几种组合 同源和FLP介导的重组产生靶向缺失 大基因组区域并产生一个自治的Episome 作为一种互惠的多方面方法的具体起源 染色体内外复制起源的分析 应阐明起源功能所需的最小区域，以及 使暴徒能够对这些特征进行精确的分子理解 有助于起源功能和启动DNA复制。