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的最重要步骤，因为它确保复制发生在细胞周期的S阶段，并且所有DNA区域都更准确地复制。起始取决于被称为起源的DNA区域的激活。这些区域通过为组装介导DNA合成的多蛋白络合物组装靶位点来提高DNA复制的效率。因此，定义起源活性所需的DNA元素对于我们对DNA复制的调节的理解至关重要。在某些单细胞真核系统中，尤其是酿酒酵母的酵母菌，已经解剖了一些起源区域，并且发现模块化元素在这些区域的功能中很重要，作为DNA复制的启动位点。相比之下，缺乏后生动物中相似区域的表征。因此，关于哪些DNA元件在DNA复制的启动步骤以及是否利用独特区域中起作用的问题仍有待回答。然而，有更多的证据表明，在真核生物染色体中的DNA复制开始与核基质有关。我们以前的工作导致了人类DNA片段的分离和克隆，这些片段与HeLa核苷酸的限制酶消化后保持与核基质有关。该DNA群体被证明富含分叉的DNA结构，以及允许在酵母菌酿酒酵母中复制的惰性质粒的序列。对这些克隆之一的分析ARSH1表明，显示出酵母细胞中这种起源活性的最小区域包含与最佳特征性酵母复制剂ARS1相似的模块化元素。更有趣的是，最近的初步数据表明，当通过电穿孔将含Arsh1的质粒转染到HeLa细胞时，能够复制。基于这些观察结果，该研究建议的主要目标是：（i）表征HeLa细胞中含有ARSH1的质粒的复制活性； （ii）研究体内染色体中ARSH1区域的复制活性； （iii）确定ARSH1区域与蛋白质的体内关联，被认为参与DNA复制的开始； （iv）识别和表征其他与ARSH1相关的HELA DNA片段的复制活性。可以预料，这些研究将更好地描述人类的复制原件，并提高我们对导致特定复制子激活的机制的了解。对这些区域的理解对于在正常和异常生长细胞中对DNA功能的牢固掌握至关重要。