DNA REPLICATION, CELL CYCLE CONTROL AND GENOME FLUIDITY

本提案的长期目标是了解 DNA复制的起始步骤受到调控。以前的研究 研究了基因扩增的分子机制， 在正常的体细胞中不发生的染色体重排 细胞，但发生在许多临床上重要的人类癌症。一个“泡沫- 从这些研究中发展出基因扩增的“断裂”模型， 假设在代谢限制下进入5相 条件通过染色体断裂促进扩增子的形成 在缓慢或停滞的复制气泡中。提出了扩增子 能够自主复制大的染色体片段或小的 片段如双微体染色体（DM）或它们的附加体 前体这项工作进一步强调，需要确定 其中DNA复制启动，并开发功能测定 来进行分子解剖 前两个具体目标建议识别，隔离， 表征使双向DNA 复制启动。三个假定的起始区域是 在过去的资助期内从附加体中鉴定和克隆。他们 不能使转染序列自主复制，但它们 在基因组中发挥作用。这就提出了起源于 功能可能取决于发生在一个 染色体一种用于起源活性的生物化学测定，和一种用于起源活性的分子生物学测定。 FLP是一种酵母位点特异性重组酶， 在哺乳动物细胞中的功能，将用于测试这一想法。此外，委员会认为， 一个起始区似乎含有复制叉屏障， 将使用生化和遗传策略进行分析。 第三个具体目标是研究结构-功能 在它们的天然染色体中复制起点的关系 位置.这是一个必须实现的基本目标，因为我们最终必须 有一种方法可以研究在原点附近的序列 对原产地活动的影响。该策略采用同源重组 在前两个图中所示的区域产生特定的改变， 具体目标对原产地功能很重要。这些研究报告将 在候选人的半合子细胞系中进行 使用几种新方法之一来删除特定的 哺乳动物基因组的区域。一种方法涉及以下组合： 同源和FLP介导重组以产生靶向缺失 并产生一个自主的附加体， 特定起源作为互惠的多方面方法 分析染色体内外的复制起点 应阐明起源功能所需的最小区域， 使我们能够从分子上精确地了解这些特征， 有助于起始功能和DNA复制的启动。

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.