This proposal is centered on regulatory controls for DNA replication in the macronucleus of the heterotrich, Euplotes. DNA replication in the macronucleus of this protozoan is highly localized into an easily-visualized replication band. The primary working hypothesis is that posttranslational modifications of specific histones are required for both the activation and inactivation of the replication process. The experiments focus on the phosphorylation of histone H1 at specific residues as necessary facets of both activation and inactivation of replication. Acetylation of histones H3 and H4 may also be important regulatory steps in the process. Replication origins will be sought in the telomeric regions of the chromosomes. These experiments will contribute to our overall understanding of DNA replication. The replication of DNA in eukaryotic cells is usually initiated at multiple sites along each of the chromosomes. Euplotes is a protozoan that exhibits an unusual level of spatial order in the pattern of DNA replication in its macronucleus, where all replication occurs in a small region. This high order in the replication pattern makes this organism particularly amenable for studies of the process. In this study, experiments are focused on regulatory aspects of DNA replication. Experiments will test whether the phosphorylation and acetylation of specific histones, at specific sites are controlling factors in the replication process. Other experiments are directed at testing whether the replication origins reside exclusively at the telomeric ends of the chromosomes. This proposal will contribute to our overall understanding of DNA replication.
