Browsing by Subject "Nucleotide excision repair"

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    Repair of DNA-protein crosslinks in mammalian cells
    (2018-07) Chesner, Lisa
    The work below describes a new assay called strand-specific primer extension-quantitative polymerase chain reaction (SSPE-qPCR) used to study the repair of DNA-protein crosslinks in mammalian cells. DNA-protein crosslinks (DPCs) are bulky lesions which disrupt important cell processes such as transcription and replication. They are formed by endogenous molecules such as formaldehyde and exogenous damaging agents such as ionizing radiation. However, the repair mechanisms associated with their repair are still unclear. Chapter 1 of this document provides background information on the formation, biological consequences, current models, and methods used to study DPC repair. Chapter 2 describes the SSPE-qPCR assay and its uses/limitations for studying the repair of plasmids containing DPCs or other polymerase-blocking adducts transfected into mammalian cells. Chapter 3 describes results generated using this assay to assess the role of nucleotide excision repair in DPC repair and highlights the versatility of the SSPE-qPCR assay. Chapter 4 extends observations made in Chapter 3 by using SSPE-qPCR to examine repair of DPC-containing plasmids in the presence of a homologous donor. It also provides evidence for homologous recombinational repair of DPCs in mammalian mitochondria. Overall, this work provides additional insight into the mechanisms of DPC repair in the nucleus and mitochondria using a quantitative, flexible assay that has not been available previously.
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    A role for UV-B -induced DNA damage in photomorphogenic responses in etiolated Arabidopsis seedlings
    (2014-01) Biever, Jessica Jo
    Ultraviolet (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.