PUTATIVE HUMAN ORIGINS OF DNA REPLICATION

DNA复制的启动是这一过程调控中最重要的一步，因为它确保复制发生在细胞周期的S期，并且所有DNA区域都能更精确地复制。启动依赖于被称为起源的DNA区域的激活。这些区域通过为介导DNA合成的多蛋白质复合物的组装提供靶位点来提高DNA复制的效率。因此，定义所需的DNA元件的起源活动是我们理解的DNA复制的调控的核心。在一些单细胞真核系统中，特别是酿酒酵母，一些起源区域已被解剖，并发现模块化元件在这些区域作为DNA复制起始位点的功能中是重要的。相比之下，后生动物生物体中的类似区域的特征缺乏。因此，什么样的DNA元件在DNA复制的起始步骤中起作用，以及是否利用了独特的区域，这些问题仍然有待回答。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。我们以前的工作已经导致了人DNA片段的分离和克隆，这些片段在限制酶消化HeLa核苷酸后仍然与核基质相关。该DNA群体被证明富含分叉的DNA结构和允许其他惰性质粒在酵母酿酒酵母中复制的序列。对这些克隆之一ARSH 1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最佳表征的酵母复制子ARS 1中存在的那些相似的模块化元件。更有趣的是，最近的初步数据表明，含有ARSH 1的质粒能够通过电穿孔转染到HeLa细胞中进行复制。基于这些观察，本研究计划的主要目标是：（i）表征含ARSH 1质粒在HeLa细胞中的复制活性;（ii）研究ARSH 1区域在体内染色体中的复制活性;（iii）确定ARSH 1区域与被认为参与DNA复制起始的蛋白质的体内关联;（iv）确定ARSH 1区域与DNA复制起始的蛋白质的体内关联。和（iv）鉴定和表征其它ARSH 1相关的HeLa DNA片段的复制活性。预计这些研究将提供一个更好的描述人类复制的原件，并增加我们的知识的机制，导致特定的复制子的激活。了解这些领域对于更好地掌握正常和异常生长细胞中的DNA功能至关重要。

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.