Experiments in this grant focus on sites used for initiation of chromosomal DNA replication using the lower eukaryote S. cerevisiae as a model system. ARSs (autonomously replicating sequences) are putative initiation sites for DNA replication recognized by their ability to support the self-replication of plasmid DNAs. The behavior of 14 different ARSs will be examined during mitosis and meiosis. For the mitotic studies, the presence and copy number of ARS plasmids will be monitored by a visual assay sensitive to plasmid copy number. These studies will provide a detailed description of the efficiency with which an individual ARS promotes the inheritance of plasmid DNAs during mitosis and meiosis. It will also be determined if trans-acting factors encoded by 2 Mum DNA can effect the mitotic stability of either synthetic or authentic yeast chromosomes. DNA contained within small replication bubbles and therefore, by definition, containing an origin of DNA replication, will be isolated from yeast chromosomal DNA after selective labelling with 4-thiouridine. Single-stranded DNA containing thio-substituted residues will be purified by affinity chromatography and used to screen a library of yeast DNA. If this origin-enriched DNA is enriched in ARSs, the experiment will establish a correlation between ARSs and replication origins in yeast chromosomes. Regardless of the outcome in terms of ARSs, the procedure will be valuable for isolating early replicating sequences which can be used in nuclear matrix experiments. The influence of nuclear sub-structure on the organization of yeast DNA, especially in terms of DNA replication, will be examined. Low salt extraction procedures will be developed to test if association of replication forks with the nuclear matrix is observed with different isolation methods. The interaction of replication origins with the nuclear matrix will be examined in populations of cells arrested at different points in the cell cycle: these experiments will determine if origins are permanently or transiently associated with the matrix. As a longer range goal, the association of other structural DNAs with the matrix will be examined. The experiments in this proposal will provide insights into mechanisms involved in control of entry into S-phase and the establishment of a temporal program for chromosome replication. Detailed knowledge of these events is important for understanding how they can be perturbed in abnormal cells.
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