Browsing by Subject "DNA Repair"
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Item Identification and characterization of novel factors that influence minisatellite stability in stationary phase yeast cells(2012-11) Alver, Bonnie MaureenThe eukaryotic genome is primarily comprised of non-coding regions of DNA consisting of several different types of repetitive elements. Minisatellites are a type of tandem repetitive element and are composed of repeat units that are 15-100bp in length. Rare altered alleles of minisatellites are associated with an increased risk of several different types of disease including cancer, diabetes, epilepsy and coronary artery disease. However, little is known about what factors prevent minisatellites from alternating and becoming potential pathogenic alleles. Our lab previously developed a color segregation assay to detect minisatellite instability in the yeast saccharomyces cerevisiae. Using this assay, we discovered a unique color segregation phenotype known as `blebbing' which was shown to be indicative of minisatellite alterations that occurred in stationary phase yeast cells. Here, we perform a genome-wide screen known as the Synthetic Genetic Array (SGA) analysis to screen for mutants strains bearing different types of minisatellite alleles that produced a strong blebbing phenotype. Through our work, we identify over 100 candidate genes that regulate the stability of a minisatellite in stationary phase. Further characterization of specific subsets of these genes demonstrates that minisatellites are regulated by different factors depending upon the repeat unit composition and size. We also demonstrate the checkpoint and mismatch repair components are important for stationary phase minisatellite stability and that alterations occurring in mutant strains are mediated by mechanisms utilizing recombination. Together our work provides novel insight into the factors governing minisatellite stability in a unique population of non-dividing cells.Item Identification and characterization of novel factors that influence minisatellite stability in stationary phase yeast cells(2012-11) Alver, Bonnie MaureenThe eukaryotic genome is primarily comprised of non-coding regions of DNA consisting of several different types of repetitive elements. Minisatellites are a type of tandem repetitive element and are composed of repeat units that are 15-100bp in length. Rare altered alleles of minisatellites are associated with an increased risk of several different types of disease including cancer, diabetes, epilepsy and coronary artery disease. However, little is known about what factors prevent minisatellites from alternating and becoming potential pathogenic alleles. Our lab previously developed a color segregation assay to detect minisatellite instability in the yeast Saccharomyces cerevisiae. Using this assay, we discovered a unique color segregation phenotype known as `blebbing' which was shown to be indicative of minisatellite alterations that occurred in stationary phase yeast cells. Here, we perform a genome-wide screen known as the Synthetic Genetic Array (SGA) analysis to screen for mutants strains bearing different types of minisatellite alleles that produced a strong blebbing phenotype. Through our work, we identify over 100 candidate genes that regulate the stability of a minisatellite in stationary phase. Further characterization of specific subsets of these genes demonstrates that minisatellites are regulated by different factors depending upon the repeat unit composition and size. We also demonstrate the checkpoint and mismatch repair components are important for stationary phase minisatellite stability and that alterations occurring in mutant strains are mediated by mechanisms utilizing recombination. Together our work provides novel insight into the factors governing minisatellite stability in a unique population of non-dividing cells.Item A role for UV-B -induced DNA damage in photomorphogenic responses in etiolated Arabidopsis seedlings(2014-01) Biever, Jessica JoUltraviolet (UV) radiation is a constituent of sunlight that influences plant morphology and growth. It induces photomorphogenic responses but also causes damage to DNA. Plant responses to DNA damage caused by UV-B light are often categorized as general mechanisms that get activated by other environmental stresses. Photodimers are formed through the direct absorption of UV-B light by DNA and are removed, in part, by nucleotide excision repair (NER). UV-B irradiation resulted in the accumulation of the two most common photodimers, cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6,4)-pyrimidinone dimers (6,4PPs), in etiolated wild type (wt) Arabidopsis seedlings. Arabidopsis mutants of the endonucleases that function in NER, xpf-3 and uvr1-1, show hypersensitivity to UV-B (280-320 nm) in terms of hypocotyl growth inhibition. I hypothesized that the accumulation of UV-B-induced photodimers was responsible for the hypocotyl growth phenotype of these NER mutants after UV-B irradiation. It was also predicted that the accumulation of photodimers could ultimately trigger signaling pathways that result in cell-cycle arrest through stalled replication sites or double-strand breaks. This was tested using the suppressor of gamma 1 (sog1-1) mutant, which lacks a transcription factor responsible for gene induction and cell-cycle arrest after gamma irradiation, and a Col-0 line containing a CYCB1;1-GUS reporter construct. CYCB1;1 encodes a cyclin that accumulates in response to cell-cycle arrest at the G2/M transition. The main conclusion from this work is that hypocotyl growth inhibition induced by UV-B light in etiolated Arabidopsis seedlings, which is a classic photomorphogenic response, is influenced by signals originating from UV-B light absorption by DNA that lead to cell-cycle arrest. Furthermore, this process is shown to occur independently of UVR8 and its signaling pathway responsible for CHS induction. This work also demonstrates that UV-B-induced DNA damage can be responsible for specific photomorphogenic responses, at least in etiolated Arabidopsis seedlings, and does not simply induce general stress responses.