Ira Leffak (Advisor)
Doctor of Philosophy (PhD)
Duplication of the genome during S phase of the mitotic cell cycle begins at thousands of sites along chromosomes termed origins of replication. Although many of the essential protein components catalyzing events at these sites are known and are conserved throughout eukaryotes, the likelihood or efficiency of initiation of DNA synthesis at any given genomic site is expected to be influenced by other novel factors, including aspects of chromatin and DNA structure.
Here I show that increased histone H4 acetylation at replication origin loci occurs after treatment with the histone deacetylase inhibitor TSA and coincides with a loss of specific initiation site selection both within origin loci and throughout the genome. Furthermore, new replication initiation sites become activated or used with greater frequency after treatment with TSA, and TSA promotes the activation of replication origins earlier during the S phase of the cell cycle. These data suggest a physiological role for histone acetylation in controlling the initiation of DNA synthesis at specific chromosomal sites.
Regions of helically unstable DNA termed DNA unwinding elements (DUEs) are commonly found at replication origins, and our laboratory identified a DUE-binding protein (DUE-B) using the c-myc DUE in a yeast one-hybrid screen. Here I demonstrate that DUE-B is required for efficient entry into S phase in human cells and for efficient replication in the Xenopus egg extract replication system. Structural analyses show the N-terminal portion of the protein to be identical to that of bacterial D-aminoacyl-tRNA deacylases. Human DUE-B possesses this function in vitro and the ability to hydrolyze ATP, suggesting that DUE-B may be a multifunctional esterase. Unique to vertebrate homologs of DUE-B is a C-terminal extension of 62 amino acids that binds DNA and is targeted for phosphorylation by CK2. The addition of the C-terminal domain to DUE-B in higher eukaryotes may have coincided during evolution with the development of a novel function for this protein in the initiation of DNA replication. Together these two sets of data argue that previously uncharacterized factors regulate the initiation of DNA replication in higher eukaryotes, possibly to deal with the complex chromosomal architecture found in these organisms.
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