STRUCTURE AND ACTIVITY OF DNA REPLICATION ORIGINS

DNA复制起点的结构和活性

• 批准号: 2734454
• 负责人: DAVID KOWALSKI
• 金额: $ 24.47万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734454
• 关键词: DNA footprinting; DNA replication origin; Saccharomyces cerevisiae; chromosomes; fungal genetics; gene mutation; genetic regulatory element; molecular genetics; nucleic acid structure; plasmids; site directed mutagenesis

The nature of DNA replication origins in eukaryotic chromosomes needs to be defined before we can understand how origin activation is specified and controlled in living cells. A commitment to activate origins in cells is, at the same time, a commitment to undergo cell division and proliferation. An understanding of the mechanisms that control origin activation is essential for a full understanding of the regulation of cell proliferation and the regulatory defects that lead to uncontrolled proliferation, as in cancer. At present, chromosomally-derived origins are best defined in the yeast, S. cerevisiae, where they have been characterized as autonomously replicating sequences (ARS) in plasmids. However, little is known about the determinants of origin activity within cellular chromosomes. In this proposal, two ARS elements have been selected for further study based on their unique properties in terms of origin function within yeast chromosomes: (l) the rDNA ARS, present in each of the 100-200 ribosomal DNA repeats, only a minority of which are actually used as replication origins in the chromosome, and (2) ARS3O3, a functional ARS element in a plasmid which is silent as an origin in its native chromosomal location. We will address two major questions of general importance to the understanding of the nature of chromosomal origins and the factors that regulate their usage: (1) what is the molecular basis for the low frequency of rDNA origin usage in a chromosome? (2) what is the molecular basis for origin silencing at ARS303 in the chromosome? To address these questions, we will (a) identify the cis-acting components that are sufficient to account for the full activity of the rDNA ADS and ARS3O3 within a plasmid, (b) identify the cis-acting elements, both positive and negative, as well as other factors that govern the level of replication origin usage in a chromosome (rDNA) and the silencing of an origin within a chromosome (ARS303) and, (c) identify DNA-protein interactions and DNA structural alterations that occur in active and silent origins, and identify the cis-components required for those interactions and structural alterations. Identification of the genetic requirements for chromosomal origin function will be achieved by taking advantage of the facility with which yeast undergo homologous DNA recombination, permitting the precise replacement of a wild-type chromosomal locus with a site-specific mutant locus carried on a plasmid vector. The proposed studies are made feasible as a result of novel approaches we have devised to assay a single-copy rDNA ARS in the chromosome and to detect origin activity associated with the normally silent ARS3O3.

真核染色体中DNA复制起源的性质需要 在我们了解如何指定原点激活之前定义 在活细胞中控制。激活细胞中起源的承诺是 同时，致力于进行细胞分裂和扩散。 对控制起源激活的机制的理解是 完全了解细胞增殖的调节至关重要 以及导致不受控制的扩散的调节缺陷，如 癌症。目前，染色体衍生的起源最好在 酵母菌，S。cerevisiae，它们被认为是自主的 质粒中的复制序列（ARS）。但是，对 细胞染色体中起源活性的决定因素。在这个 提案，已经选择了两个ARS元素，以进一步研究 它们在酵母中起源功能方面的独特属性 染色体：（l）rDNA ARS，存在于100-200核糖体中的每一个中 DNA重复，只有少数被用作复制 起源于染色体，（2）ARS3O3，一个功能性ARS元素 质粒在其天然染色体位置静音为起源。 我们将解决两个主要重要性的主要问题 了解染色体起源的性质及其因素 调节其用法：（1）低的分子基础是什么 染色体中rDNA起源的使用频率？ （2）什么是分子 染色体中ARS303的原点沉默的基础？解决这些 问题，我们将（a）确定是顺式作用组件 足以说明RDNA ADS和ARS3O3的全部活动 在质粒中，（b）确定阳性和 负面的负面因素，这些因素控制了复制水平 起源在染色体（rDNA）中的使用和在内部的沉默 染色体（ARS303）和（c）鉴定DNA蛋白质相互作用和DNA 在主动和沉默的起源中发生的结构变化，以及 确定这些相互作用和结构所需的顺式组件 改变。鉴定染色体的遗传需求 来源功能将通过利用设施与 哪种酵母菌经历同源DNA重组，允许精确 用位点特异性突变体代替野生型染色体基因座 位于质粒载体上的基因座。提出的研究是可行的 由于新的方法，我们已经设计出来分析单拷贝 染色体中的rDNA ARS并检测与 通常沉默的ARS3O3。