PUTATIVE HUMAN ORIGINS OF DNA REPLICATION

启动DNA复制是这一过程调控中最重要的步骤，因为它确保复制发生在细胞周期的S阶段，并且所有DNA区域复制得更精确。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体的组装提供靶点来提高DNA复制的效率。因此，确定起始活动所需的DNA元件对于我们理解DNA复制的调控是至关重要的。在一些单细胞真核系统中，特别是酿酒酵母中，一些起始区已经被解剖，并发现模块元件在这些区域作为DNA复制的起始点的功能中是重要的。相比之下，缺乏后生动物生物中类似区域的特征。因此，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，这一问题仍有待回答。然而，越来越多的证据表明，真核细胞染色体中DNA复制的启动与核基质有关。我们以前的工作导致了人类DNA片段的分离和克隆，这些片段在限制性内切酶消化HeLa核苷酸后仍与核基质相关。这一DNA群体被证明富含分叉的DNA结构和允许惰性质粒在酿酒酵母中复制的序列。对其中一个克隆ARSH1的分析表明，在酵母细胞中显示这一起源活性的最小区域包含与特征最好的酵母复制子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，当通过电穿孔将含有ARSH1的质粒导入HeLa细胞时，它们能够复制。基于这些观察，本研究的主要目标是：(I)鉴定含有ARSH1的质粒在HeLa细胞中的复制活性；(Ii)研究ARSH1区域在活体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联系；以及(Iv)鉴定和表征其他与ARSH1相关的HeLa DNA片段的复制活性。预计这些研究将提供对人类原始复制的更好描述，并增加我们对导致特定复制子激活的机制的了解。对这些区域的了解对于更坚定地掌握正常和异常生长细胞中的DNA功能是必不可少的。

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.