PUTATIVE HUMAN ORIGINS OF DNA REPLICATION

DNA复制的起始是调控这一过程中最重要的一步，因为它确保复制发生在细胞周期的S期，并且所有DNA区域复制得更加精确。起始依赖于被称为起始的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点来提高DNA复制的效率。因此，定义起源活性所需的DNA元素是我们理解DNA复制调控的核心。在一些单细胞真核生物系统中，特别是酵母，一些起源区域已经被解剖，并发现模块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，后生生物中类似区域的特征是缺乏的。因此，哪些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。我们之前的工作已经导致分离和克隆人类DNA片段，这些片段在HeLa核苷酸的限制性内切酶消化后仍然与核基质相关。该DNA群体被证明具有丰富的分叉DNA结构和序列，这些序列允许其他惰性质粒在酵母中复制。对其中一个克隆ARSH1的分析表明，酵母细胞中显示这种起源活性的最小区域包含与最具特征的酵母复制子ARS1中存在的模块化元素相似。更有趣的是，最近的初步数据表明，当通过电穿孔转染到HeLa细胞中时，含有arsh1的质粒能够复制。基于这些观察结果，本研究计划的主要目标是：(i)表征含有arsh1的质粒在HeLa细胞中的复制活性；（ii）研究ARSH1区域在体内染色体中的复制活性；（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）鉴定和表征其他与arsh1相关的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原始，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.