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.

In the past few years, cytoplasmic processing bodies (P-Bodies) have been identified in eukaryotic cells. P-bodies have roles in translational repression, mRNA storage, mRNA decay and are conserved cytoplasmic aggregations of non-translating mRNAs in conjunction with translation repression and mRNA degradation factors. In this work, I, in collaboration with others provide evidence for a new biological role for P-bodies in viral life cycles. This work can be summarized thus:In a collaborative effort, I have identified connections between retrovirallike transposon life cycles and P-bodies. For example, genetic evidence in yeast indicates that key proteins within P-bodies are required for the life cycles of the Ty1 and Ty3 retrotransposons. Moreover, Ty3 genomic RNA (gRNA) as well as viral structural proteins accumulate in P-bodies, suggesting that P-bodies may serve as sites of viral assembly.Second, I have shown, with assistance of collaborators, that the positivestrand RNA virus, Brome Mosaic Virus (BMV) gRNA accumulates in P-bodies Moreover, viral RNA dependent RNA polymerase (RdRp) colocalizes with and co-immunoprecipitates with the P-body protein Lsm1p, suggesting that P-bodies may participate in viral replication. Remarkably, the accumulation BMV gRNA in P-bodies is dependent on cis-elements that have been demonstrated to play critical roles in viral RNA replication.The identification of P-bodies as sites of accumulation of viral gRNA and viral proteins of both retro-virus like elements and positive-stranded RNA viruses, expands the list of important biological roles played by P-bodies. Since P-body proteins and structure are highly conserved, these findings imply that Pbodies will be important for other RNA viruses.

Mouse Hepatitis virus (MHV) is a member of the coronavirus family in the order Nidovirales. The 32 kb genome contains cis-acting sequences necessary for replication of the viral genome. Those cis-acting sequences have been shown to bind host proteins, and binding of those proteins is necessary for virus replication. One of the cis-acting elements is the 3' terminal 42 nucleotide host protein binding element. Previous work has demonstrated that mitochondrial aconitase, mitochondrial heat shock protein 70, heat shock protein 60 and heat shock protein 40 bind to the 3' terminal 42 nucleotide host protein binding element. We demonstrated that RNA secondary structure of the 3' terminal 42 nucleotide host protein binding element is necessary for host protein binding in vitro. We also demonstrate that primary structure of the 3' terminal 42 nucleotide host protein binding element is necessary for viral replication by targeted recombination. DI replication assays infer that the 3' terminal 42 nucleotide host protein binding element plays a role in positive strand synthesis from the negative strand template. Current studies involve the infectious cDNA clone, which will provide definitive answers on the role of the 3' terminal 42 nucleotide host protein binding element in MHV replication.

In Saccharomyces cerevisiae, DNA replication initiates at distinct origins termed Autonomously Replicating Sequences (ARSs). A key element in ARS is the ACS/B1 sequence, which binds the Origin Recognition Complex (ORC). During early G1 phase, the pre-replication complex is assembled by ORC. These ARSs are termed replicators. In yeast, ORC is also involved in gene silencing. These loci also contain an ACS/B1 element; however at these positions ORC recruits the silencing SIR complex. ARSs found here are termed silencers. Therefore, ARSs have a dual function. Research has also shown that origin ARSs can be substituted for silencing ARSs and vice versa. Since a mutation in ACS abolishes replication activity, studies have turned to the B1 element to account for the functional duality, but results have been mixed. I hypothesize that the B1 element plays a key role in the dual nature of ARS. To test this hypothesis, silencer and replicator ARSs were subjected to site directed mutagenesis around the WTW motif of the B1 element. Their efficiency was then tested using routine silencing and replication assays. Results reveal that the silencing ability of silencer ARSs is unaffected by these mutations; however mutations within and around the WTW motif reduce silencing efficiency of replicator ARSs. Sequence alignments have also shown that silencer ARSs have a broader WTW consensus than replicator ARSs. Preliminary replication assays are consistent with the above results and other research, contributing to a conclusion that the B1 element is not the sole determining factor in the dual nature of ARS. / NSERC

DNA damage tolerance (DDT) is a process utilized by cells to bypass replication blocking lesions in the DNA, preventing replication fork collapse and maintaining genomic stability and cell viability. In Saccharomyces cerevisiae DDT consists of two branched pathways. One branch allows direct replication past lesions in the DNA utilizing specific error-prone polymerases, a process known as translesion DNA synthesis (TLS). The other branch utilizes homologous recombination and template switch to replicate past damaged DNA in an error-free manner. RAD5 has traditionally been characterized as belonging to the error-free pathway of DNA damage tolerance. The protein is multi-functional, with several specific activities identified and classified to the error-free branch of DDT. However, there is also evidence for additional uncharacterized activities of the protein. The goal of this research was to determine which branches of DNA damage tolerance the uncharacterized activities of Rad5 are involved in. A two-pronged approach was utilized, elucidation of the physical interactions of the protein, and examination of the genetic interactions between RAD5 and other DDT genes. The evidence indicates that Rad5 plays a partial role in TLS and the protein is known to physically interact with Rev1, a member of the TLS pathway. We assumed this physical interaction mediates the TLS activity of Rad5. The yeast two-hybrid assay was utilized to examine the interaction between Rev1 and truncated Rad5 fragments, and the N-terminal 30 amino acids of Rad5 proved sufficient to maintain the interaction. This research sets the stage to identify key residues in Rad5 for the interaction with Rev1, and the creation of a TLS deficient rad5 mutant by targeting those key residues. Genetic interactions between RAD5 and genes required for the initiation of DDT in the cell were examined based on sensitivity to killing by various DNA damaging agents. We determined that the functions of Rad5 rely on PCNA modification, and thus do not function in a cellular process unrelated to Rad5. Potential uncharacterized functions are discussed on the basis of these results and the results of the interaction studies. Future structural and functional studies are proposed to better understand the role of Rad5 in the cell.

The advancement in computing in the last three decades has introduced many devices in our daily lives including personal computers, laptops, cellular devices and many more. The data we need for our processing needs is scattered among these devices. The availability of all the scattered data in the devices in use associated to an individual user as one is achieved in a Personal Information Environment. Data recharging is a technique used to achieve a Personal Information Environment for an individual user using data replication. In this thesis, we propose a data recharging scheme for an individual user’s Personal Information Environment. We study the data availability to a user by conducting a simulation using the data recharging algorithm. This data recharging approach is achieved by using master-slave data replication technique. / Thesis (Master, Computing) -- Queen's University, 2009-08-06 00:18:00.19

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.