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Measuring, Modeling and Identifying Factors That
Influence Eukaryotic DNA Replication
by
Simon Robert Vincent Knott
A Dissertation Presented to the
Faculty Of The USC Graduate School
University Of Southern California
In Partial Ful llment of the
Requirements for the Degree
Doctor Of Philosophy
(Computational Biology and Bioinformatics)
May 2011
Copyright 2011 Simon Robert Vincent Knott
Object Description
| Title | Measuring, modeling and identifying factors that influence eukaryotic DNA replication |
| Author | Knott, Simon Robert Vincent |
| Author email | knott@usc.edu; srvknott22@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Computational Biology and Bioinformatics |
| School | College of Letters, Arts and Sciences |
| Date defended/completed | 2011-03-28 |
| Date submitted | 2011 |
| Restricted until | Restricted until 09 Nov. 2011. |
| Date published | 2011-11-09 |
| Advisor (committee chair) | Tavaré, Simon |
| Advisor (committee member) |
Smith, Andrew Laird, Peter Aparicio, Oscar |
| Abstract | For cells to proliferate, the genome must be replicated exactly once per cell cycle in a timely and accurate manner. Making this task difficult are multiple other genomic processes, such as transcription and DNA repair, that are concurrently operating on the same genomic template. Replication initiates at specific loci called replication origins that must undergo a series of protein loadings before they can begin to replicate. Although this loading schedule takes place at all origins, individual origins fire at distinct and conserved times during S-phase. It has been suggested that origin firing schedules are defined by their propensity to attract rate limiting replication factors from limited pools (where origins with higher propensities replicate earlier and origins with lower propensities replicate later). This model has not been validated and, furthermore, the factors determining an origin's propensity to attract replication factors remains poorly understood. In higher eukaryotes, replication timing has been linked to epigenetic inheritance and genomic stability. Thus, determining which factors dictate origin timing schedules is important for understanding the mechanisms driving development and healthy cell proliferation.; This current work investigates the molecular kinetics that drive the S. cerevisiae replication schedule and also begins to uncover what coordination they exhibit with concurrently operating genomic processes. To understand better these timing dynamics, we begin by developing molecular and computational tools to analyze replication timing genome-wide. Using these tools we produce a novel dataset that represents the highest fidelity temporal map of DNA replication to date. Next, to identify novel candidate limiting factors to DNA replication, we describe and analyze (in the context of this temporal map) two additional datasets designed to capture both pre-S-phase replication protein loading and global origin efficiencies.; Through analysis of these data we determine that, in G1-phase, the earliest replicating origins show a high propensity to attract Cdc45 (a replication factor that is limited in its nuclear concentration G1-phase). Following this, we devise and computationally implement a detailed theoretical model of DNA replication to test the hypothesis that origin firing dynamics (and hence genome-wide replication times) are determined by their ability to recruit replication factors from limited pools. After validating this model we identify factors that, in unperturbed cells, are correlated with origin firing dynamics. These include nucleosome positioning around the origin and the clustering of origins in the nucleus in late G1-phase.; Previous work has demonstrated that histone acetylation around an origin promotes its early replication. Specifically, others have shown that when the histone-deacetylase Rpd3 is removed from the cell several origins increase in their activity. To test the scope of Rpd3's action at origins, we have analyzed Rpd3 mutant cells genome-wide for their origin replication activities. We determined that approximately one-third of origins are suppressed by Rpd3 action. By targeting the individual complexes that Rpd3 operates in, we determined that its action at origins is through its role in transcriptional repression at the gene promoter, as opposed to its broad action as a suppressor of spurious transcription events. Furthermore, we demonstrate that the regions surrounding Rpd3-regulated origns are deacetylated by Rpd3 and also that these regions are enriched for Rpd3 binding and Rpd3-regulated genes.; Finally, we introduce the forkhead transcription factors Fkh1 and Fkh2 as two novel regulators of origin function. We demonstrate that Fkh1 alone regulates ~50 origins and that in cells where both Fkh1 and Fkh2 action is removed, over one-half of all origins show deregulation. Furthermore, these factors are the first to be identified that have both repressive and excitatory action at origins (~100 origins are activated by Fkh1 and Fkh2 while ~80 are repressed; Fkh-excited and -repressed, respectively). As mentioned above, Cdc45 association at origins in G1-phase is predictive of their function. We demonstrate that in fkh1Δfkh2Δ cells, this factor is depleted at Fkh-excited origins. Furthermore, we demonstrate that Fkh-excited origins are not found near the centromere, in contrast Fkh-repressed origins include many origins that localize at the centromere. Finally, we determine that Fkh1 and Fkh2 likely have their action at origins by regulating the formation of long-range chromatin interactions. Furthermore, we show evidence suggesting (that to regulate these interactions) individual forkhead proteins bind at different origins and then dimerize to bring these origins together in the nucleus. |
| Keyword | replication origin; modeling; transcription; chromatin |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m3931 |
| Rights | Knott, Simon Robert Vincent |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Knott-4521 |
| Archival file | uscthesesreloadpub_Volume48/etd-Knott-4521.pdf |
Description
| Title | Page 1 |
| Full text | Measuring, Modeling and Identifying Factors That Influence Eukaryotic DNA Replication by Simon Robert Vincent Knott A Dissertation Presented to the Faculty Of The USC Graduate School University Of Southern California In Partial Ful llment of the Requirements for the Degree Doctor Of Philosophy (Computational Biology and Bioinformatics) May 2011 Copyright 2011 Simon Robert Vincent Knott |
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