PUTATIVE HUMAN ORIGINS OF DNA REPLICATION

DNA 复制的假定人类起源

• 批准号: 6349122
• 负责人: MANUEL SEVERO VALENZUELA
• 金额: $ 11.77万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349122
• 关键词: DNA replication origin; HeLa cells; Saccharomyces cerevisiae; fungal genetics; genetic mapping; nucleic acid sequence; plasmids

Initiation of DNA replication is the most important step in the regulation of this process since it ensures that replication occurs during the S phase of the cell cycle and that all DNA regions replicate precisely more. Initiation depends on the activation of DNA regions known as origins. These regions increase the efficiency of DNA replication by provid9ing target sites for the assembly of multi-protein complexes that mediate DNA synthesis. Therefore, defining the DNA elements required for origin activity is central to our understanding of the regulation of DNA replication. In some single-cell eukaryotic systems notably, the yeast Saccharomyces cerevisiae, some origin regions have been dissected and modular elements found to be important in the functioning of these regions as initiation sites for DNA replication. In contrast, the characterization of similar regions in metazoa organisms is lacking. Thus, question as to what DNA elements play a role in the initiation step of DNA replication, and whether unique regions are utilized, remain to be answered. There is increased evidence however, that initiation of DNA replication in eukaryote chromosomes is associated with the nuclear matrix. Our previous work has led to the isolation and cloning of human DNA fragments which remain associated to the nuclear matrix after restriction enzyme digestion of HeLa nucleotides. This DNA population was shown to be enriched for forked DNA structures and for sequences that allow an otherwise inert plasmid to replicate in the yeast Saccharomyces cerevisiae. Analysis of one of these clones, ARSH1, showed that the minimal region showing this origin activity in yeast cells, contains modular elements similar to those present in the best characterized yeast replicator, ARS1. More interestingly, recent preliminary data suggests that ARSH1-containing plasmids are able to replicate when transfected into HeLa cells by electroporation. Based on these observations, the major goals of this research proposal are: (i) to characterize the replication activity of ARSH1-containing plasmids in HeLa cells; (ii) to investigate the replication activity of the ARSH1 region in vivo chromosomes; (iii) to determine the in vivo association of the ARSH1 region to proteins that are thought to participate in the initiation of DNA replication; and (iv) identify and characterize the replication activity of other ARSH1-related HeLa DNA fragments. It is anticipated that these studies will provide a better description of human originals of replication and increase our knowledge about the mechanisms that leads to the activation of specific replicons. An understanding of these areas is essential for a firmer grasp of DNA function in normal and abnormal growing cells.

DNA 复制的启动是调节该过程中最重要的步骤，因为它确保复制发生在细胞周期的 S 期，并且所有 DNA 区域的复制更加精确。启动取决于被称为起源的 DNA 区域的激活。这些区域通过为介导 DNA 合成的多蛋白复合物的组装提供靶位点来提高 DNA 复制的效率。因此，定义起始活性所需的 DNA 元件对于我们理解 DNA 复制的调控至关重要。在一些单细胞真核系统中，特别是在酿酒酵母中，一些起源区域已被解剖，并且发现模块化元件对于这些区域作为DNA复制起始位点的功能非常重要。相反，缺乏后生动物中类似区域的表征。因此，关于哪些 DNA 元件在 DNA 复制的起始步骤中发挥作用以及是否利用了独特的区域等问题仍有待解答。然而，越来越多的证据表明，真核生物染色体中 DNA 复制的起始与核基质有关。我们之前的工作已经分离和克隆了人类 DNA 片段，这些片段在限制性内切酶消化 HeLa 核苷酸后仍与核基质相关。该 DNA 群体被证明富含分叉 DNA 结构和序列，这些序列允许惰性质粒在酿酒酵母中复制。对这些克隆之一 ARSH1 的分析表明，在酵母细胞中显示这种起源活性的最小区域包含与特征最明确的酵母复制子 ARS1 中存在的模块元件相似的模块元件。更有趣的是，最近的初步数据表明，含有 ARSH1 的质粒在通过电穿孔转染到 HeLa 细胞中时能够复制。基于这些观察，本研究计划的主要目标是：（i）表征 HeLa 细胞中含有 ARSH1 的质粒的复制活性； (ii) 研究ARSH1区域在体内染色体的复制活性； (iii) 确定 ARSH1 区域与被认为参与 DNA 复制起始的蛋白质的体内关联； (iv) 鉴定并表征其他 ARSH1 相关 HeLa DNA 片段的复制活性。预计这些研究将更好地描述人类复制的起源，并增加我们对导致特定复制子激活的机制的了解。了解这些区域对于更牢固地掌握正常和异常生长细胞中的 DNA 功能至关重要。