PUTATIVE HUMAN ORIGINS OF DNA REPLICATION

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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.