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This project addressed mechanisms of the neuronal DNA damage response after treatment with the model DNA damaging agent ultraviolet light (UV). The thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU) protected against UV-initiated neuronal apoptosis in a concentration-dependent manner (p<0.001). BrdU did not protect proliferating mouse embryonic fibroblasts from UV-induced apoptosis. We assessed whether the mechanism of BrdU neuroprotection was through a modification in the neuronal DNA damage response. BrdU neuroprotection was independent of BrdU incorporation into DNA, neuronal DNA repair, p53 activation or cell cycle re-entry, a neuronal DNA damage response. Neurons deficient in Cockayne Syndrome B (CSB) or Xeroderma Pigmentosum A (XPA) were paradoxically resistant to UV-initiated apoptosis. Therefore, CSB and XPA play essential roles in the neuronal DNA damage response.

This project addressed mechanisms of the neuronal DNA damage response after treatment with the model DNA damaging agent ultraviolet light (UV). The thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU) protected against UV-initiated neuronal apoptosis in a concentration-dependent manner (p<0.001). BrdU did not protect proliferating mouse embryonic fibroblasts from UV-induced apoptosis. We assessed whether the mechanism of BrdU neuroprotection was through a modification in the neuronal DNA damage response. BrdU neuroprotection was independent of BrdU incorporation into DNA, neuronal DNA repair, p53 activation or cell cycle re-entry, a neuronal DNA damage response. Neurons deficient in Cockayne Syndrome B (CSB) or Xeroderma Pigmentosum A (XPA) were paradoxically resistant to UV-initiated apoptosis. Therefore, CSB and XPA play essential roles in the neuronal DNA damage response.

The gene that produces the precursor RNA transcript to the three largest ribosomal RNA molecules (rDNA) is present in multiple copies and organized into gene clusters. They represent 0.5% of the diploid human genome but are critical for cellular viability. The individual genes possess very high levels of sequence identity and are present in high local concentration, making them ideal substrates for genomic rearrangement driven by dysregulated homologous recombination. Our laboratory has developed a sensitive physical assay capable of detecting recombination-mediated genomic restructuring in the rDNA by monitoring changes in lengths of the individual clusters. In order to determine whether dysregulated recombination is a potential driving force of genomic instability in human cancer, adult patients with either lung or colorectal cancer, and pediatric patients with leukemia were prospectively recruited and assayed. Over half of the adult solid tumors show detectable rDNA rearrangements relative to either surrounding non-tumor tissue or normal peripheral blood. In contrast, there is a greatly reduced frequency of alteration in pediatric leukemia. This finding makes rDNA restructuring one of the most common chromosomal alterations in adult solid tumors, illustrates the dynamic plasticity of the human genome, and may have prognostic or predictive value in disease progression.

Methylmercury (MeHg) is an environmental toxin to which we are exposed through the consumption of seafood. Reactive oxygen species (ROS) have been implicated in the mechanism of toxicity, and in vitro studies in our laboratory have implicated DNA oxidation, particularly the DNA repair enzyme oxoguanine glycosylase 1 (OGG1). My studies determined the effects of in utero exposure to MeHg on fetal brain DNA oxidation and postnatal neurodevelopmental deficits, and the role of ROS-mediated oxidative DNA damage using the free radical spin trap, α-phenyl-N-tert-butylnitrone (PBN), and DNA repair-deficient ogg1 knockout mice. While neither MeHg nor PBN altered DNA oxidation in fetal brain, MeHg caused cognitive deficits in passive avoidance and novel object recognition, the latter of which was blocked by PBN pretreatment, suggesting ROS involvement. Preliminary longevity studies following one litter from each treatment group to 16 months suggest that in utero MeHg treatment may shorten lifespan. Endogenous DNA oxidation was increased in the brains of ogg1 knockout fetuses compared to wild-type littermates, although this was not enhanced by MeHg. However, OGG1-deficient animals exhibited cognitive deficits in passive avoidance after MeHg treatment, suggesting a role for DNA damage. Furthermore, ogg1 knockout female mice exhibited a passive avoidance deficit compared to wild-type females regardless of treatment, corroborating a role for oxidative DNA damage in neurodevelopmental deficits. MeHg increased apoptosis in the hippocampal region of fetal brain, and may cause DNA double-strand breaks (DSBs), evidenced by enhanced phosphorylation of histone 2AX (γH2AX). Ogg1 knockout progeny exhibited increased cellular proliferation or migration in the developing hippocampal region, which was blocked by MeHg. My results provide the first evidence that: (1) MeHg may decrease lifespan; (2) PBN protects against some postnatal neurodevelopmental deficits caused by in utero exposure to MeHg; and (3) DNA repair-deficient progeny are more susceptible to postnatal cognitive deficits caused by in utero MeHg exposure, suggesting that ROS-mediated DNA oxidation plays a role in MeHg-initiated neurodevelopmental deficits.

Evaluating the evolutionary role of mutations depends on an understanding of their major properties, including their rate of origin, U, and the distribution of their fitness effects, f(s). While substantial effort has been put into measuring these properties, most studies have only examined their distributions in a single context. In nature, spontaneous mutations are likely to experience heterogeneity in genetic and environmental context, and this could lead to variation in both U and f(s). My thesis investigates the changes in U and f(s) with different genetic and environmental factors in Drosophila melanogaster, in order to elucidate the nature of context-associated variation in mutational properties. Examination of condition-dependent variation in DNA repair showed that high and low conditioned individuals differ in the use of alternative repair pathways. This could ultimately lead to variance in their heritable mutation rates. However, the assumption that condition dependence in repair arises solely due to a presumed trade-off between accuracy and the energetic costs associated with different repair pathways is too simplistic. Instead, physiological considerations appear to mediate condition-dependent changes in DNA repair. Measurements of selection on individual mutations across different genetic and environment contexts showed that context-associated changes in mutational fitness effects are common. I found that heterogeneity in fitness effects across different environments result in changes to the overall mean and variance of f(s). This does not, however, seem attributable to the degree of ‘adaptedness’ of a population to a particular environment (a prediction generated by previous theoretical analysis). On the other hand, f(s) appears to be relatively robust to differences among genotypes, with epistasis averaging close to zero. This finding suggests that genetic and environmental perturbations may affect mutations differently. Overall, my thesis represents the most rigorous empirical investigation to date of the conceptual and theoretical predictions regarding the nature of context-dependent heterogeneity in U and f(s) for multicellular eukaryotes.

Radiation therapy targets cancer cell death by overwhelming cells with harmful DNA damage. Understanding how cells repair radiation damage and in particular how they become resistant to radiation therapy is important for effective cancer treatment. Our lab made the novel discovery that Bcl10, a cytoplasmic protein important for NF-B activation, localizes to endogenous H2AX foci in the nucleus of breast cancer cells. We determined that following radiation treatment Bcl10 is recruited to ionizing radiation-induced foci in a dose-dependent matter and that it is important for the repair of radiation-induced DNA damage. We also observed that breast cancer cells are extremely sensitive to Bcl10 knockdown, causing cellular senescence, while normal breast epithelial cells are insensitive. Our findings identify Bcl10 as potent anti-cancer target. / Experimental Oncology

The integrity of the genome is critical to organisms and it is affected by many factors. Radiation, for example, poses a serious threat to genome stability of human beings. While physical monitors for radiation hazard are present, the biological consequences of long term exposure to radiation are not well understood. With the opportunity as part of the International Caenorhabditis elegans Experiment-1 flight project, several approaches using C. elegans were taken to measure mutational changes that occurred during the spaceflight. Among these methods, the eT1 balancer system was demonstrated to be well-suited as an integrating biological dosimeter for spaceflight. The dog-1 gene in C. elegans is required to prevent mutations at poly-G/poly-C tracts, and previous work has described that in the absence of DOG-1, small deletions initiate within these tracts, most likely as a consequence of improperly repaired replication blocks. The eT1 balancer system was adapted to investigate the broad mutational spectrum of dog-1 mutants. Using this system, I was able to determine a forward mutation rate of approximately 1 x 10-3, 10 fold higher than spontaneous. Both small deletions as reported previously and unreported large chromosome rearrangements were observed, and most of mutations analyzed are associated with G/C tracts. Thus, I propose that following dog-1-induced replication blocks, repair leads to a wide range of mutational events and chromosomal instabilities, similar to those seen in human cancers. The existence of the G/C tracts in C. elegans creates a fortuitous but perplexing problem. They are hotspots for genome instability and need enzymatic protection. In the genome of C. elegans, approximately 400 G/C tracts exist and are distributed along every chromosome in a non-random pattern. G/C tracts are also over-represented in another Caenorhabditis species, C. briggsae. However, the positions and distribution differ from those in C. elegans. Furthermore, in C. elegans, analysis of SAGE data showed that the position of the G/C tracts correlated with the level of gene expression. Although being a threat to genome stability, the genomic distribution of G/C tracts in C. elegans and their effect on regional transcription levels suggest a role for G/C tracts in chromatin structure.

Thesis (Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (leaves 167-182). Also available via World Wide Web.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80-87). Also available for download via the World Wide Web; free to University of Oregon users.