Cells of ataxia telangiectasia (AT) individuals are hypersensitive to a variety of DNA damaging agents such as ionizing radiation and bleomycin, presumed to be due to an intrinsic defect in repair of DNA damage. The nature of the DNA lesion(s) to which AT cells are abnormally sensitive, and the defect in DNA repair are presently unclear. The major part of this project aimed at investigating the sensitivity of AT cells to DNA double-strand breaks (dsb) generated by restriction endonucleases (RE), thereby verifying the hypothesis that AT cells are deficient in the processing of dsb. AT lymphoblastoid cell lines (AT-PA and AT-KM) used in this study were initially characterized and found to be approximately 3 times more sensitive to ionizing radiation in the induction of micronuclei (Mn) and chromosomal aberrations (CA) compared with a normal lymphoblastoid cell line (N-SW). Other cellular characteristics were observed in AT-PA cells following-irradiation such as normal induction and rejoining of dsb and reduced inhibition of DNA synthesis. By using SLO poration, RE were introduced into the AT and normal cell lines and the yield of CA resulting from RE-induced dsb were subsequently investigated. The frequencies of CA induced by Pvu II were 2 - 4 fold higher in AT-PA than in N-SW cells at both 5 h and 24 h sampling times. The enhanced frequency of CA in AT cells treated with Pvu II was principally a result of an increase of chromatid aberrations, rather than chromosome aberrations at 24 h. higher frequencies of chromatid exchanges appeared in AT-PA than in N-SW cells. The results suggest that AT cells are characterized by a defect in dsb processing that converts a higher number of dsb into CA than in the normal cell line. With respect to the different end-structures of RE-induced dsb, cohesive-ended dsb generated by BamH I and Pst I were found to induce lower frequencies of CA than blunt-ended dsb generated by Pvu II and EcoR V in both the AT cell lines and the normal cell line. The results support the previous observations that cohesive-ended dsb are less clastogenic than blunt-ended dsb (Bryant 1984). Although inducing lower frequencies of CA than Pvu II and EcoR V, BamH I and Pst I induced higher number of CA in both AT-PA and AT-KM cells when compared with N-SW cells, again indicating a defect in processing cohesive-ended dsb exists in AT cells. A potent DNA repair inhibitor, Ara A, was found to potentiate the production of CA by RE in AT and normal cells. The enhancement ratios (by ara A) for CA induced by Pvu II and Pst 1 were higher in N-SW cells than in AT-PA and AT-KM cells. Ara A appeared to have no effect on the frequencies of CA induced by BamH I in any of the cell lines tested. Based on these findings, a mechanism for the rejoining of RE-induced dsb in which DNA repair synthesis may be involved is proposed, and it is postulated that dsb in AT cells are subjected to greater end degradation. Inhibition of DNA synthesis was observed in normal cells after treatment with Pvu II and EcoR V, while EcoR I and BamH I had only minor effect. AT-PA cells were found to be resistant to RE-induced inhibition of DNA synthesis, as in the case of ionizing radiation. This result suggests that RE-induced blunt-ended dsb mimic radiation-induced lesions in supressing DNA synthesis in normal cells and that AT cells respond to RE-induced dsb in a similar way to damage induced by ionizing radiation. Finally, when a nuclear extract from N-SW cells was introduced into Pvu Il-treated AT-PA cells, it was able to confer a normal frequency of CA. In contrast, neither whole cell nor nuclear extracts from normal cells influenced the production of CA induced by y-rays. These findings provide evidence for the presence of factor(s) in normal nuclear extract which complements the defect in processing of RE-induced dsb in AT cells.

Mutations are generally thought to be targeted events. The distribution of mutations is based upon the initial original deposition of DNA damage and the fidelity and efficiency of repair of this damage. These factors are dependent on the primary site of DNA modification and the surrounding nucleotides (i.e., mutation is “context sensitive"). To better understand mutagenesis, I measured DNA damage and/or mutation at the DNA sequence level, then considered the impact of mutation location and the surrounding nucleotide environment. The selected mutagens, ultraviolet light (UV) and benzo(a)pyrene [B(a)P], were chosen because they produce well-characterized lesions and are environmentally relevant. UVC (254nm) light-induced DNA damage is well documented. UVC produces a characteristic spectrum of mutations. The predominant UV-induced mutations are C → T transitions occurring at TC or CC sites, as well as CC → TT tandem transitions. The latter class of mutation is considered the hallmark of UV mutagenesis. Quantitatively speaking, the primary types of UV-induced DNA lesions are cyclobutane pyrimidine dimers (CPDs) and the 6-4 pyrimidine/pyrimidone (64PyPy). These are also the suspected predominant pre-mutagenic lesions. Each lesion was independently measured at the DNA sequence level in a defined region of DNA. The pattern of UVC-induced DNA damage revealed a complex induction pattern. The flanking DNA nucleotides partially influenced the pattern of damage deposition. Sites where C → T transitions and CC → TT transitions were recovered at high frequencies were also frequently damaged. Thus, at these sites, mutation fixation was potentially more influenced by initial DNA damage than the rate of DNA repair. Two other components of the UV spectrum [UVB (290-320 nm) and UVA (320-400 nm)] are more environmentally relevant than UVC since UVB and UVA reach the surface of the earth.The results of UVC experiments were used as a guide to interpret the results obtained using UVB since direct light absorption by DNA has been shown to be one of the main biological effect at both wavelengths. The model that was chosen for the studies was an in vivo transgenic rodent mutagenesis assay. The research presented in the thesis represents one of the first studies to characterize UV-induced mutation at the DNA sequence level in rodent skin. The backs of female C57B1/6 lacl transgenic mice were shaved and exposed to either UVB or UVA light. UVB was found to be significantly more mutagenic than UVA. The UVB-induced mutation spectrum was characterized by C → T transitions at dipyrimidine sites, implicating CPD and/or 64PyPy lesions as premutational DNA lesions. The majority of UVA-induced mutations was C → T transitions at dipyrimidines sites and hence, as with UVB-induced mutation, attributed to CPDs and 64PyPy. In the UVA-dose response experiments, the induced mutant frequency was lower than expected at higher doses. A statistically significant increase in putative clonal expansion suggested that skin cells might have undergone cell killing followed by repopulation. In a final study, C57Bl/6 lacI transgenic male mice were intraperitoneally injected with the mutagen B(a)P at doses of 0, 62.5, 125, 250, and 500 mg/kg. This resulted in a linear increase in mutation frequency (4.8 to 53 × 10⁻⁵). All mutations increased at GC basepairs and not AT basepairs following B(a)P treatment. This was consistent with models suggesting guanosine adducts to be mutagenic lesions. In conclusion, the transgenic lacI mouse mutagenesis model was a sensitive target for in vivo mutagenesis from UVB, UVA and benzo(a)pyrene exposures. The system detected class-specific mutation frequency differences and increases in cell proliferation after mutagen exposure. With a further refinement of techniques, the correlation of DNA damage and mutation will allow even more exquisite studies. / Graduate

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Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / It has been demonstrated that oligonucleotides homologous to the 3' telomere repeat sequence TTAGGG (T-oligos) stimulate DNA damage responses that are also induced by disruption of the telomere loop structure. Adaptive defense against oxidative stress and UV or ionizing radiation has been reported, but adaptive antioxidant defense as a response to mimicking telomere loop exposure has not been described. The T-oligos pTT and pGTTAGGGTTAG were added to human dermal fibroblast cultures to investigate whether mimicking telomere loop disruption stimulates antioxidant defense. pTT stimulated mitochondrial superoxide dismutase protein levels within 72 hours. Cell yields were higher after H202 exposure in fibroblasts pretreated with pTT for 72 hours compared to diluent pretreated cells. Intracellular reactive oxygen species (ROS) levels, measured by flow cytometry and the dichlorofluorescein diacetate probe, increased during T-oligo treatment as compared to diluent and oligonucleotide controls. The time course and degree of ROS stimulation corresponded to the time course for activation and/or induction of p53 and p21/Cip1/Waf1. The NADPH oxidase inhibitor diphenyliodonium chloride abrogated this increase and fibroblasts retrovirally transduced to produce dominant negative p53 failed to display increased ROS, implicating that the T-oligos induced ROS through p53-responsive NADPH oxidases. A horseradish peroxidase assay for extracellular H20 2 showed no H20 2 release with pTT treatment. To determine whether there was induction of senescence, an endpoint response to increased ROS and prolonged T-oligo treatment in fibroblasts, the senescence-associated β-galactosidase assay was conducted in parallel with the DCF assay. Only the 11mer T-oligo treatment modestly increased the number of β-galactosidase positive cells by 72 hours (<30% of cells). This is the first report suggesting that antioxidant defense and ROS signaling are part of the broad adaptive response in mammalian cells presumably initiated by telomere loop disruption and mimicked by T-oligos. T-oligo treatment thus offers a new model for studies of ROS signaling in human dermal fibroblasts, allowing exploration of the relationships between DNA damage, ROS, oxidative stress, and the evolution of cellular defense mechanisms. / 2031-01-01

Thesis (Ph. D.)--School of Dentistry and School of Pharmacy. University of Missouri--Kansas City, 2004. / "A dissertation in oral biology and pharmacology." Advisor: J. David Eick. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Feb. 22, 2006. Includes bibliographical references (leaves 176-189). Online version of the print edition.

Huntington’s disease is an autosomal dominantly inherited neurodegenerative disorder characterized by degeneration of striatal and cortical neurons. The neurons in these regions are particularly energy-demanding and need to maintain high levels of oxidative phosphorylation to support cellular activities. Reactive oxygen species are generated as a byproduct of oxidative phosphorylation and can damage DNA and other biomolecules if not properly metabolized. In HD, there is elevated oxidative DNA damage and impaired DNA damage repair, likely due to impaired function of the mutant huntingtin protein in base excision repair (BER). Previous studies have shown that mutant huntingtin is hypo-phosphorylated at serines 13 and 16 in the N17 domain, and that restoring phosphorylation can reestablish normal protein function and is protective in HD. In this thesis, we show that a metabolite of the DNA damage product N6- furfuryladenine (N6FFA), kinetin triphoshate (KTP) increases N17 phosphorylation through casein kinase 2 (CK2) by acting as an ATP analog, with protective effects in cell and animal models of disease. We additionally show N6FFA increases the activity of CK2 on other substrates, specifically p53. We hypothesize that in times of ATP stress CK2 can utilize KTP as an alternate energy source, promoting DNA repair and cell viability. In HD, inefficient BER inhibits generation of KTP and promotes hypo- phosphorylation of CK2 substrates, which can be overcome by exogenous addition of N6FFA. Additionally, we show that another DNA-responsive kinase, PKCζ, can also phosphorylated N17, potentially priming this domain for CK2 phosphorylation. Finally, we propose that the protective effects of N6FFA may be via a two-pronged pathway, involving both CK2 and the mitochondrial quality control kinase, PINK1. Thus, this thesis presents a novel mechanism where a product of DNA damage acts as a phosphate source for critical kinases in DNA repair and mitochondrial maintenance in conditions where ATP levels are low. / Thesis / Doctor of Philosophy (PhD)

Chromosomal rearrangements can lead to loss of heterozygosity (LOH) and oncogene activation, both of which represent possible causative events in cancer development. Such outcomes can result from the misrepair of DNA damage arising from a variety of events including DNA double-strand breaks (DSBs), collapsed replication forks, and dysfunctional telomeres. In response to a DSB, chromosomal stability is principally maintained through the two major DNA repair pathways; non- homologous DNA end-joining (NHEJ) and homologous recombination (HR). The objective of this thesis was to identify novel factors functioning in prevention of chromosomal instability in response to a DSB in Schizosaccharomyces pombe. To achieve this, a central aim was to identify the genes mutated in a number of radiation-sensitive mutants in fission yeast, previously isolated by the laboratory. These include the ‘loh’ mutants loh-2, loh-5, loh-6 and loh-7, which were found to harbour mutations in known DNA repair genes rad3, rad17, and rad57. Further, a pan-genomic screen for novel HR repair factors was carried out. The Bioneer Version 2 deletion-library, consisting of 3308 haploid deletion strains, was screened for strains displaying hypersensitivity to the DNA damaging agents MMS, bleomycin and camptothecin. This screen yielded 209 hits which were further characterised, utilising a set of non-essential Ch¹⁶ minichromosomes . The minichromosome Ch¹⁶-LMYAU harbours an HO endonuclease recognition sequence and a centromere-distal ade6-M216 heteroallele. Following break-induction, failed repair of the DSB leads to loss of the ade6 allele, indicated by pink sectoring on low adenine plates. 39 sectoring hits were identified and further characterised to quantify levels of gene conversion via HR in response to a DSB, utilising Ch¹⁶-RMYAH. As a result of this study, a group of novel genes functioning in HR repair were identified. Finally, one of these hits, putative RNA metabolism protein Nrl1, was subjected to further characterisation, associating this protein with DNA damage repair for the first time. The work presented here, documents the approaches taken to successfully identify novel DNA repair factors in fission yeast.

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