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Regulation of DNA Replication Initiation by Histone Acetylation and the DNA Unwinding Element Binding Protein DUE-B

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Regulation of DNA Replication Initiation by Histone Acetylation and the DNA Unwinding Element Binding Protein DUE-B Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2006 Regulation of DNA Replication Initiation by Histone Acetylation and the DNA Unwinding Element Binding Protein DUE-B Michael George Kemp Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Biomedical Engineering and Bioengineering Commons Repository Citation Kemp, Michael George, "Regulation of DNA Replication Initiation by Histone Acetylation and the DNA Unwinding Element Binding Protein DUE-B" (2006). Browse all Theses and Dissertations. 41. https://corescholar.libraries.wright.edu/etd_all/41 This Dissertation is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. REGULATION OF DNA REPLICATION INITIATON BY HISTONE ACETYLATION AND THE DNA UNWINDING ELEMENT BINDING PROTEIN DUE-B A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy By MICHAEL G. KEMP B.S., Wright State University, 1999 _____________________________ 2006 Wright State University i COPYRIGHT MICHAEL G. KEMP 2006 ii WRIGHT STATE UNIVERSITY SCHOOL OF GRADUATE STUDIES August 21, 2006 I HEREBY RECOMMEND THAT THE DISSERTATION PREPARED UNDER MY SUPERVISION BY Michael G. Kemp ENTITLED Regulation of DNA Replication Initiation by Histone Acetylation and the DNA Unwinding Element Binding Protein DUE-B BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy. _________________________ Michael Leffak, Ph.D. Dissertation Director _________________________ Gerald Alter, Ph.D. Director, Biomedical Sciences Ph.D. Program _________________________ Joseph F. Thomas, Jr., Ph.D. Dean, School of Graduate Studies Committee on Final Examination _________________________ Steven Berberich, Ph.D. _________________________ Thomas L. Brown, Ph.D. _________________________ Patrick Dennis, Ph.D. _________________________ Mark Mamrack, Ph.D. iii ABSTRACT Kemp, Michael G. Ph.D., Biomedical Sciences Program, Wright State University, 2006. Regulation of DNA Replication Initiation by Histone Acetylation and the DNA Unwinding Element Binding Protein DUE-B. 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 iv 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. v TABLE OF CONTENTS Page INTRODUCTION...........................................................................................................................................1 Licensing the replication origin..................................................................................................................1 Activating the replication origin.................................................................................................................6 Initiation of DNA synthesis........................................................................................................................9 Replicator elements in eukaryotes............................................................................................................10 Unicellular replicators .........................................................................................................................10 Embryonic systems..............................................................................................................................11 Xenopus egg extracts ...........................................................................................................................11 Mammalian replication origins............................................................................................................12 A role for novel factors at replicators.......................................................................................................15 A role for chromatin structure and histone acetylation in DNA replication ........................................15 DNA unwinding elements ...................................................................................................................17 MATERIALS AND METHODS ..................................................................................................................19 Cell culture ...............................................................................................................................................19 Cell synchronization.................................................................................................................................19 Nascent strand abundance assays .............................................................................................................19 Preparation of formaldehyde-crosslinked chromatin................................................................................20 Quantitative PCR......................................................................................................................................21 Random sequence primer PCR.................................................................................................................21 Western blotting .......................................................................................................................................22 Flow cytometry.........................................................................................................................................22 Cell Fractionation.....................................................................................................................................23 EMSA.......................................................................................................................................................24 Transfections – siRNA/DNA....................................................................................................................24 Cloning of DUE-B for expression in human cells....................................................................................25 Generation of recombinant DUE-B baculoviruses ...................................................................................26 vi TABLE OF CONTENTS (continued) Purification of baculovirus-expressed rDUE-B........................................................................................27 Purification of rDUE-BHeLa.......................................................................................................................27 Preparation of Xenopus egg extracts.........................................................................................................28 Immunodepletion of proteins from Xenopus egg extracts ........................................................................30 Xenopus sperm chromatin replication assay.............................................................................................31 Sperm chromatin spin-down experiments ................................................................................................31 Limited proteolysis...................................................................................................................................31 D-amino acid deacylase assay ..................................................................................................................32
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