Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 169-183).

The efficiency of metazoan origins of DNA replication is known to be enhanced by histone acetylation near origins. Although this correlates with increased MCM recruitment, the mechanism by which such acetylation regulates MCM loading is unknown. We show here that Cdt1 induces large-scale chromatin decondensation that is required for MCM recruitment. This process occurs in G1, is suppressed by Geminin, and requires HBO1 HAT activity and histone H4 modifications. HDAC11, which binds Cdt1 and replication origins during S-phase, potently inhibits Cdt1-induced chromatin unfolding and re-replication, suppresses MCM loading, and binds Cdt1 more efficiently in the presence of Geminin. We also demonstrate that chromatin at endogenous origins is more accessible in G1 relative to S-phase. These results provide evidence that histone acetylation promotes MCM loading via enhanced chromatin accessibility. This process is regulated positively by Cdt1 and HBO1 in G1 and repressed by Geminin-HDAC11 association with Cdt1 in S-phase, and represents a novel form of replication licensing control.

DNA replication is a fundamental cellular process. However, the structure and dynamics of the eukaryotic DNA replication machinery remain poorly understood. A soluble extract system prepared from Xenopus eggs recapitulates eukaryotic DNA replication outside of a cell on a variety of DNA templates. This system has been used to reveal many aspects of DNA replication using a variety of ensemble biochemical techniques. Single-molecule fluorescence imaging is a powerful tool to dissect biochemical mechanisms. By immobilizing or confining a substrate, its interaction with individual, soluble, fluorescently-labeled reactants can be imaged over time and without the need for synchrony. These molecular movies reveal binding parameters of the reactant and any population heterogeneity. Moreover, if the experiments are imaged in wide-field format, the location or motion of the labeled species along the substrate can be followed with nanometer accuracy. This dissertation describes the use and development of novel single-molecule fluorescence imaging techniques to study eukaryotic DNA replication. A biophysical characterization of a replication fork protein, PCNA, revealed both helical and non-helical sliding modes along DNA. Previous experiments demonstrate that the egg extracts efficiently replicate surface-immobilized linear DNA. This finding suggested replication of DNA could be followed as motion of the replication fork along the extended DNA. However, individual proteins bound at the replication fork could not be visualized in the wide-field due to the background from the high concentration of the fluorescent protein needed to compete with the extract’s endogenous protein. To overcome this concentration barrier, I have developed a wide-field technique that enables sensitive detection of single molecules at micromolar concentrations of the labeled protein of interest. The acronym for this method, PhADE, denotes three essential steps: (1) Localized PhotoActivation of fluorescence at the immobilized substrate, (2) Diffusion of unbound fluorescent molecules to reduce the background and (3) Excitation and imaging of the substrate-bound molecules. PhADE imaging of flap endonuclease I (Fen1) during replication revealed the time-evolved pattern of replication initiation, elongation and termination and the kinetics of Fen1 exchange during Okazaki fragment maturation. In the future, PhADE will enable the elucidation of the dynamic events at the eukaryotic DNA replication fork. PhADE will also be broadly applicable to the investigation of other complex biochemical process and low affinity interactions. It will be especially useful to those researchers wishing to correlate motion with binding events.

published_or_final_version / Paediatrics / Master / Master of Philosophy

Chromosomal fragile sites are specific loci associated with a high frequency of breakage and recombination. A cell's ability to repair and/or replicate through a lesion is prerequisite to the maintenance of genomic stability. An improved understanding of fragile site biology and its contribution to replication defects and genomic instability is critical for prevention, intervention, and diagnosis of genetic diseases such as cancer. This work seeks to identify and characterize both trans and cis fragile sites associated elements involved in instability onset and progression. An array of Saccharomyces cerevisiae isogenic DNA repair deficient mutants were utilized to identify genes contributing to the stability or instability of a natural fragile site ~ 403 kb from the left telomere on chromosome VII. Findings suggest that the RAD52 epistasis group, the MRX complex, non-homologous end-joining (NHEJ) pathways, MUS81 and SGS1 helicases, translesion polymerases, and a majority of the post replication repair (PRR) proteins are all required for faithful replication of the 403 fragile site and likely other fragile sites as well. In contrast I found that MMS2, previously thought to be specific to the PRR pathway, is required to prevent the fusion of repetitive elements within the 403 site. mgs1 (homolog of the human Werner helicase interacting protein, WHIP) and pol3-13 (a subunit of the DNA polymerase delta) mutants also exhibited reduced instability in checkpoint deficient cells. These findings suggest previously uncharacterized function of Mgs1, Pol3 and Mms2 in regulation of genome regions at risk of replication damage. We further find the presence of inverted repeats (IR) are sufficient to induce instability. Two IR's proximal to the 403 site consistently fuse to generate acentric and dicentric chromosomes involving the 403 fragile site and a newly identified site on chromosome VII as well. The frequency of fusion events is aggravated by chromatin traffic stressors such as tRNA transcription induced fork stalling and replisome termination regions.

Late expression factor 3 is one of the six AcMNPV genes essential for DNA replication identified through transient replication assays. LEF-3 is a single stranded DNA binding protein responsible for the transportation of the viral helicase (P143) into the nucleus of the infected cell. In this study, a protein complementation-based assay was adapted to identify the region(s) of LEF-3 that is (are) involved in LEF-3-LEF-3 protein interactions. The full-length LEF-3, or various truncated LEF-3 regions were fused with Venus1 (N- terminus portions of full length Venus, a modified yellow fluorescence protein) or Venus2 (C- terminus). Venus1 and Venus2 fragments generated a functional fluorescent Venus protein when the two fragments were brought together by protein-protein interaction of the fused LEF-3 constructs. Fluorescence generated by coexpression of full-length LEF-3 fusion proteins confirmed that LEF-3 exists as homo-oligomer. Interaction between the full-length and the N- terminal (aa 1-189) or C- terminal regions (aa 190-385), and between the various truncated LEF-3 regions suggested the complexity of LEF-3 oligomeric structure. LEF-3 constructs deleted for NLS function revealed cytoplasmic fluorescence, suggesting that LEF-3-LEF-3 interactions occur in the absence of DNA or nuclear proteins. Because LEF-3 is essential for nuclear transporting the viral helicase (P143), the ability of LEF-3 to interact with another viral protein was investigated. P47, a sub-unit of the viral RNA polymerase was chosen because it is cytoplasmic when expressed on its own. The interaction between LEF-3 and P47 produced complete nuclear localized fluorescent signals. Overall, the results suggest that there are multiple regions of LEF-3 that are capable of closely interacting, and that multiple domains are likely involved in the oligomerization of full-length LEF-3. The interaction of LEF-3 with P47 suggests that P47 may be another LEF-3 cargo protein. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-05-27 15:02:53.983

Dephosphorylation of the Cdc2 kinase by the Cdc25 tyrosine phosphatase is the universally conserved trigger for mitotic entry. Cdc25 is also the point of convergence for checkpoint signaling pathways which monitor the genome for damaged DNA and incomplete replication. In addition, Cdc25 is inhibited by a MAP kinase cascade in the event of osmotic, oxidative and/or heat stress. These pathways inhibit cell cycle progression by phosphorylating Cdc25 resulting in its association with 14-3-3 and nuclear export. Although Cdc25 can be observed leaving the nucleus following inhibitory signals it is controversial whether phosphorylation, 14-3-3 binding or export itself is required for checkpoint proficiency. In fission yeast, Cdc25 is phosphorylated in vitro on 12 serine and threonine residues by the effector kinase of the DNA replication checkpoint, Cds1. Nine of these residues reside in the N-terminal regulatory region, while three are found in the extreme C-terminus of the protein. We show here that phosphorylation the nine N-terminal residues, nor any of the 12 in vitro sites, are required for enforcement of the DNA replication checkpoint. In lieu of Cdc25 phosphorylation the phosphatase is rapidly degraded and mitotic entry prevented by the action of the Mik1 kinase, targeting Cdc2. Thus, multiple mechanisms exist for preventing mitotic entry when S-phase progression is inhibited. The three C-terminal in vitro phosphorylation sites have not previously been examined in fission yeast. However, homology exists between the S. pombe protein and the Cdc25 orthologues in humans, Xenopus and Drosophila in this region. We report here that in S. pombe these sites are required to prevent mitotic entry following replication arrest in the absence of Mik1, and in the maintenance, but not establishment, of arrest following DNA damage. Our previous work showed that Cdc25 nuclear import requires the Sal3 importin-β but at the time we were unable to show a direct interaction between these two proteins. The final chapter of this thesis proves physical interaction by co-immunoprecipitation. Cdc25 mutants lacking all twelve putative Cds1 sites show nuclear localization during mitosis in a sal3- background, effectively reversing the cell cycle regulated pattern of accumulation of the phosphatase. / Thesis (Ph.D, Biology) -- Queen's University, 2011-06-20 12:16:15.71

DNA replication is cruciaI for the transmission of genetic information. Understanding the enzymology involved in this complex process will allow further insight into its mechanism. Experimental evidence indicates a role for DNA ligase I in DNA replication. Techniques of molecular and cellular biology and immunology were utilized in this study to further investigate DNA ligase I and clarify its involvement and interaction with other proteins in DNA replication. Immunofluorescence studies were performed to examine the intracellular distribution of DNA ligase I. Confocal analysis of indirect immunofluorescence detection of DNA ligase I using affinity purified anti-human DNA ligase I antibodies showed nuclear localization of DNA ligase I in distinct foci resembling those structures seen in detection of centres of DNA replication and other DNA replication proteins. Immunoprecipitation experiments were performed on extracts of MDBK cells to examine possible interactions of DNA ligase I with the DNA replication cofactor, PCNA; and no interactions were detected.

This study examined the DNA replication timing patterns of endothelial cell (EC)-enriched genes. We especially focused on a unique set of EC-enriched mRNA transcripts that possess differentially methylated regions (DMRs) within proximal promoters. It was previously shown that this DNA methylation plays an important functional role in regulating EC-enriched patterns of gene expression. Since the maintenance of these silencing marks is necessary for the inheritance of cell identity, the cell should ensure the proper transmission of such marks during mitotic cell cycle. Here we show that EC-enriched genes with DMRs replicate early during S phase in both expressing and non-expressing cell types. EC-enriched genes that do not have DMRs followed the expected trend, being early replicating in expressing cell types and late in non-expressing cell types. The relationship between DNA replication and DNA methylation was also investigated. A delay between DNA replication and DNA methylation was observed.

This study examined the DNA replication timing patterns of endothelial cell (EC)-enriched genes. We especially focused on a unique set of EC-enriched mRNA transcripts that possess differentially methylated regions (DMRs) within proximal promoters. It was previously shown that this DNA methylation plays an important functional role in regulating EC-enriched patterns of gene expression. Since the maintenance of these silencing marks is necessary for the inheritance of cell identity, the cell should ensure the proper transmission of such marks during mitotic cell cycle. Here we show that EC-enriched genes with DMRs replicate early during S phase in both expressing and non-expressing cell types. EC-enriched genes that do not have DMRs followed the expected trend, being early replicating in expressing cell types and late in non-expressing cell types. The relationship between DNA replication and DNA methylation was also investigated. A delay between DNA replication and DNA methylation was observed.