Michael Leffak (Advisor), Weiwen Long (Committee Member), John Paietta (Committee Member)
Master of Science (MS)
The best characterized eukaryote replication model is of the budding yeast Saccharomyces cerevisiae. Replication origins of S.cerevisiae are 100 to 200 bp in size and contain an essential 11-bp autonomous replicating sequence (ARS) consensus sequence (ACS). The origin recognition complex (ORC) binds to the ACS in order to recruit additional replication factors (Cdt1, Cdc6, MCM, Cdc45) and together they form the pre-replication complex (pre-RC).
Unlike budding yeast, the mammalian cells contain dispersed replication origins in which multiple elements distributed over large distances act as replication start sites. Mammalian DNA replication origins, such as the c-myc origin, contain a DNA unwinding element (DUE), which is an AT-rich region that contains10-of-11 matches to ARS consensus. Our lab primarily studies DNA replication of the human c-myc locus. We have successfully integrated the wild-type 2.4 kb c-myc replication origin and its various inactive mutants in a known ectopic chromosomal site in HeLa/406 cells in order to study the multiple functional elements of this origin.
My thesis focused on investigating the minimal sequence required in mammalian replication origin for replication activity. To address this question, I assessed the effects of replication protein tethering on chromatin accessibility of inactivated c-myc origins which are 930 bp or less and contain an intact DUE. In part I of my thesis, I describe how I made two new HeLa/406 cell lines containing two different deletion mutants of the cmyc replicator core (which are 607 bp and 228 bp long) at the known ectopic chromosomal site. In part II of my thesis, I describe how I created plasmids expressing GAL4 DNA-binding domain (GAL4DBD)-BRG1 fusion proteins. BRG1 is a catalytic core subunit of SWI/SNF mammalian chromatin remodeling complex. Wild-type BRG1 and catalytic inactive BRG1K798R mutant were used to make GAL4DBD fusion proteins.
In part III of my thesis, I evaluated the effects of GAL4DBD fusion proteins on chromatin structure of the inactivated ectopic c-myc replicator via DNase I hypersensitivity assay. The proteins used were Cdt1, a licensing protein necessary for pre-RC formation; wild-type BRG1 and catalytically dead BRG1 mutant. GAL4DBD-Cdt1 has previously been shown to restore origin activity, as well as binding of replication proteins such as ORC, MCM, Cdc45, and DUE-B. My data suggest that expression of GAL4DBD-Cdt1 or of GAL4DBD-wild-type BRG1 can significantly increase the DNase I sensitivity of the inactivated ectopic 5' 930 bp c-myc origin, which contains both the DUE and the GAL4 binding sites. However, the shorter c-myc replicators (607 bp or 228 bp) did not show any change in DNase I hypersensitivity when the same GAL4DBD-Cdt1 was expressed. These results indicate that in conjunction with GAL4DBD protein tethering, the c-myc DUE alone is not sufficient for opening the chromatin structure of the ectopic c-myc replicator. Thus, the reactivation of the ectopic c-myc replicator via GAL4DBD protein tethering requires additional DNA elements besides the DUE.
Department or Program
Department of Biochemistry and Molecular Biology
Year Degree Awarded
Copyright 2015, all rights reserved. My ETD will be available under the "Fair Use" terms of copyright law.