No description available.

Homologous recombination (HR) is a precise mechanism for repairing harmful DNA double-strand breaks. The process has been extensively studied in microbial species leading to identification of the major proteins, HR models and homology requirements. Much less is known about HR in mammalian systems, especially early HR events. Our laboratory has recently devel-oped an assay that detects the new DNA synthesis that accompanies the early homology search and strand invasion steps of HR (the 3’ extension assay). The hypothesis that homology require-ments for the early steps of HR may differ from those identified in other HR assays was tested. Plasmids bearing varying amounts of homology to the chromosomal immunoglobulin μ target locus gene were constructed and tested in the 3’ extension assay. The homology require-ments for the 3’ extension assay were somewhat lower than might be expected based on other HR assays. An approximately linear relationship between homology length and 3’ extension was also established on each side of the double-strand break. The effect of excess Rad51, an essential protein involved in early HR, was also measured with respect to homology, leading to the dis-covery that increased Rad51 resulted in an increase in 3’ extension events independent of ho-mology. In summary, 3’ extension generates a potentially unstable, short-lived HR intermediate that has less dependence on homology than a completed HR product. Homology plays a role in the initiation of HR, but it may be more important in the stabilization of the intermediate than the actual generation of the early HR product detected in the 3’ extension assay. / CIHR

No description available.

No description available.

<p>Topoisomerase III&alpha; (Top3&alpha;) is an essential component of the double Holliday junction (dHJ) dissolvasome complex in metazoans. Previous work has shown that Top3&alpha; and Bloom's helicase (Blm) are able to convergently migrate the dHJ to create solely non-crossover products, thus preserving genomic integrity. However, many questions remain about the details of this process. Using a combination of biochemical and genetic tools, including dHJ substrate assays, gel electrophoresis, EMSA, pulldowns, fly crosses, and electron microscopy, this work expands our knowledge of the dissolution reaction. Tail mutants of Top3&alpha; were created and tested in a series of <italic>in vitro</italic> assays. Through these experiments, I discovered that the C-terminus of Top3&alpha; is important for binding Blm, interacting with DNA, conveying RPA stimulation, and <italic>in vivo</italic> functionality. I also observed that dissolution is an extremely processive reaction, with no accumulation of intermediates prior to product formation. When a non-specific topoisomerase was used (Top1, a type IB), accumulation of an intermediate was evident; however, contrary to predicted models, direct observation revealed that this intermediate is not a hemicatenane structure and still requires branch migration. Modifications were also made to the dHJ substrate creation method so that multiple types of HJ substrates could be produced efficiently.</p> / Dissertation

Holliday junctions are formed as an intermediate during DNA recombination as the two strands come together. Recombination occurs during meiosis, and also during DNA double strand repair. Trapping this branched intermediate could prevent DNA repair from occurring in cells which would prove beneficial during cancer treatment. There are many enzymes that cleave Holliday junctions. One such enzyme, T7 Endonuclease I, was specifically chosen to detect ligand binding at the core of the junction since its binding and cleavage of cruciforms is well documented. Specialized bifunctional ligands were studied in this project that were designed to bind DNA structures that are held in close proximity to one another. These compounds have two identical binding modules that are connected by a linker of various length and rigidity, with each module binding very weakly; however, when both modules bind the binding affinity is greatly enhanced. The interactions of these compounds with cruciforms are currently being studied.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24280号 / 医博第4896号 / 新制||医||1061(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 森田 智視, 教授 松田 道行, 教授 波多野 悦朗 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Successful replication of the genome during each cell cycle requires that every replication fork merge with its opposing fork. However, lesions in the template DNA or protein-DNA barriers often impede replication forks and threaten the timely completion of genome duplication. If a fork encounters a replication fork barrier (RFB), it can be subject to a variety of fates. In some cases the replisome is maintained in a manner such that it can resume DNA synthesis when the barrier is removed. Alternatively the stalled fork is simply held in a competent state to merge with the opposing fork when it arrives. However, fork stalling can also precipitate dissociation of the replisome (fork collapse) or even fork breakage. If this happens the recombination machinery can intervene to restore DNA integrity and restart replication, albeit with a risk of causing deleterious genetic change if ectopic homologous sequences are recombined. I have exploited a site-specific RFB in fission yeast termed RTS1 to investigate the consequences of perturbing a single replication fork. RTS1 is a polar RFB (i.e. it blocks fork progression in a unidirectional fashion), enabling replication to be completed by the opposing fork. Despite this, fork blockage at RTS1 triggers a strong recombinational response that is able to restart DNA synthesis, which at least initially is highly error prone. Here, I present my work in establishing a live cell imaging approach to visualizing the recombinational response at the RTS1 RFB, demonstrating that the majority of cells initiate recombination-dependent replication (RDR). RDR begins within a few minutes of fork blockage and is only curtailed by the arrival of the opposing fork. It depends on the Rad52 protein, which remains associated with the restarted fork and whose presence correlates with its infidelity. I also illustrate the significance of various genetic factors, including Rad51, the Rad51 mediators, Fml1 helicase, Rad54 translocase, Pfh1 sweepase, and Cds1 checkpoint kinase, in modulating Rad52 localization and block-induced recombination at the RTS1 RFB.

Coronaviruses have caused high pathogenic epidemics within the human population on two occasions; in 2003 a coronavirus (HCoV-SARS) caused severe acute respiratory syndrome and in 2012 a novel coronavirus emerged named Middle East respiratory syndrome (HCoV-MERS). Four other species of coronavirus circulate endemically in the human population (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1), which cause more benign respiratory disease than either HCoV-SARS or HCoV-MERS. The emergence of HCoV-MERS provides an additional opportunity to study the characteristics of coronaviruses. Reverse genetics can be used to study an organism’s phenotype by logical mutation of its genotype. Construction of an infectious clone construct provides a means to investigate the nature of HCoV-MERS by reverse genetics. An HCoV-MERS infectious cDNA clone system was constructed to use for reverse genetics by homologous recombination of bacterial artificial chromosomes (BACs). This system should aid in answering remaining questions of coronavirus genetics and evolution as well as expedite the development of vaccines and prophylactic treatments for HCoV-MERS. / October 2015