Thesis (Ph. D.)--University of Oregon, 2002. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 101-112). Also available for download via the World Wide Web; free to University of Oregon users.

The RecQ family of helicases has been termed the “Caretakers of the Genome,” and rightfully so. These proteins are highly conserved from bacteria to humans and have been implicated in functions from homologous recombinatorial repair to damage checkpoint response to telomere maintenance and more. Mutant genes of three of the human RecQ helicases lead to syndromes characterized by a high incidence of cancer, premature aging and early death. Despite their implications in several biological functions and importance to the integrity of the human genome and suppression of cancer, many aspects of the RecQ family structure and function remain unknown. To date, much is known about the catalytic function of the helicase domain and accompanying domains, but considerably less is known about the non-catalytic N-terminus in these proteins, which, in many cases, including those human orthologs involved in disease, can make up about half of the total protein length. While experiments have been able to identify protein partners that interact with the N-terminal region, few are able to narrow the binding sites to minimally functional parts and fewer still describe any detail regarding the structural features of these binding areas. In fact, some reviews have generally described the N-terminus as “featureless,” a concept we challenge in our studies. Many of the N-termini of these RecQs have long been known to contain large stretches of acidic residues, a feature of intrinsically disordered regions. These regions/proteins are rich in charged and polar residues, lack compactness that makes crystallography possible, and have flexible and dynamic conformations that are prevalent in “high specificity, low affinity” interactions. Disordered proteins are well-known to be hot spots for protein/protein interactions and post-translational modifications, amongst other functions. Considering these facts, and recognizing the ties between these and what we know about the N-termini of the RecQs, we hypothesized that these proteins likely have long disordered termini. In Chapter 3, we confirm the presence of disorder at the Top3/Rmi1 binding site on Sgs1, the Saccharomyces cerevisiae RecQ helicase. We show that even in a disordered state, this binding region is not “featureless,” but in fact contains a transient alpha-helical molecular recognition element that is necessary to facilitate complex formation between Sgs1, Top3 and Rmi1. Loss of helical structure at this site leads to increased genomic instability and sensitivity to DNA damaging agents. Based on these results, we suggest that there are likely many more such elements in the N-terminus that that are important for other Sgs1 protein/protein interactions and provide an estimate for the number of interactions in this region. In Chapter 4, we evaluate the prevalence of disorder in a set of Chromatin Processes proteins in an effort to establish a role for disorder with regards to maintaining chromatin integrity. In our bioinformatics study, we found that disorder is overrepresented in the Chromatin Processes proteins, and that a major driving force for disorder in these proteins is protein/protein interaction and post-translational modification. We also show a biological connection to disorder and increased protein/protein interaction by investigating these parameters in the context of the DNA damage checkpoint response and in complex formations. Mediators between highly structured kinases in the checkpoint were the most interactive proteins and over half of all predicted interaction sites occurred in disordered areas. Complexed proteins often contained one protein with a high number of disordered sites and a high number of predicted interactions, while the rest were considerably more ordered. Chapter 5 explores a Sgs1 interaction partner, Rmi1 and uses bioinformatics to design structurally-based point mutations in an effort to further elucidate Rmi1 function in yeast, which remains largely unknown outside of its enhancement of Top3/Sgs1 catalytic function. Using AGADIR, which predicts alpha-helical structure and is particularly useful in our hands for guided-mutagenesis in disordered regions, we identified several point mutations that lead to Δrmi1 phenotypes or intermediate growth on hydroxyurea. We hypothesize that these mutants are important in maintaining Rmi1 stability. Together, these studies suggest an important change in how the field approaches further studies into the RecQ helicases; traditional methods of primary sequence comparisons and crystal structures limit the study of disordered regions that are still functionally important. Future care should be given to consider the conservation of structure or structural elements in the RecQs over strict alignments when comparing functional regions between orthologs. Our studies also suggest that it is highly likely that structural motifs for important protein interactions in RecQs are being overlooked because they are not readily obvious using traditional methods. By understanding these motifs and the interactions they facilitate, we may be able to more easily identify polymorphisms in patients with genomically unstable conditions like cancer and, having better understood the biological process these structures facilitate, design drugs to counteract detrimental effects.

Colon cancer is the second most common type of cancer in the United States. Its incidence is linked epidemiologically to high levels of bile acids in the feces. Bile acids have been implicated as promoters and cocarcinogens in the etiology of colon cancer and as comutagens and mutagens in bacteria. These observations suggest the hypothesis that bile acids may damage DNA. By using the DNA-damage inducible SOS system in Escherichia coli, this study shows that when bacteria are exposed to bile acids there is induction of the SOS repair system and preferential survival of cells undergoing repair. Additionally, differential killing assays using repair defective bacteria show strains defective in recombinational repair or excision repair have lower survival when treated with bile acids than their parental wild-type counterparts. Human fibroblasts were treated with bile acids and unscheduled DNA synthesis (UDS) was measured. UDS is considered to represent the DNA synthesis step in excision repair. UDS, measured by autoradiography, was found to significantly increase in human fibroblasts upon treatment with bile acids. In addition, differential cytotoxicity assays with Chinese Hamster Ovary cells showed that different DNA-repair pathway defective cells were sensitive to different bile acids. Introduction of DNA damage and induction of DNA-repair by bile acids implicates them as possible direct carcinogens in the etiology of colon cancer.

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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.

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.

Loss of genomic stability is associated with a variety of diseases, particularly cancer. Of the many proteins which maintain genomic integrity, two of the most important are MLH1 and MSH2, which participate in DNA mismatch repair. Previous work established derivatives of the CaCo2 human colorectal cancer cell line with siRNA-mediated knockdown of these proteins. When xenografted into mice, tumors with reduced MLH1 or MSH2 expression grew faster than controls. Following growth in vivo, clonal cell lines were established from the tumors and used to examine the effects that knockdown of MSH2 had on other members of the DNA mismatch repair system. Clonal survival following exposure to 5-fluorouracil was also evaluated, and those cells with reduced MLH1 and MSH2 levels were found to be resistant. This study has implications for the importance of knowing the MMR status of a given tumor when deciding on a course of treatment, and of the compounding effects of the loss of one MMR protein on others in the family. / Canadian Cancer Society Research Institute

In this work we studied various aspects of DNA repair in plants, focusing mainly on point mutation repair and its interconnection with double-strand break repair. We were using transgenic point mutation and recombination substrates as a primary tool in our experiments. We have compared two transgenic homologous recombination assays (B-glucuronidase- and luciferase-based), analyzed the sensitivity of DNA repair machinery to ultraviolet radiation and assessed the involvement of AtKu80, Atm and AtXpd repair genes in point mutation repair. Ours study revealed the following: the luciferase-based recombination assay is more sensitive then B-glucuronidase-based; double-stand break repair machinery is sensitive to ultraviolet radiation, which results in increased pint mutation formation; chosen DNA repair genes might be impaired in point mutation repair, however further experimentations are needed to confirm this. / xi, 132 leaves : ill. ; 29 cm.

Oxidative stress and histone deacetylase (HDAC) inhibition has been implicated as potential mechanisms in valproic acid (VPA) teratogenicity. Reactive oxygen species (ROS) can target DNA to cause oxidative DNA damage and DNA double strand breaks (DSBs) which can be repaired through homologous recombination (HR). HR is not an error free process and can result in detrimental genetic changes. In this present study we evaluated the role of HDAC inhibition in VPA-initiated HR. HDAC inhibition may indirectly alter repair activity as a result of increased expression of genes involved in HR or indirectly by causing DNA damage which initiates repair. The first objective was to investigate the ability of VPA to cause HDAC inhibition in the Chinese hamster ovary (CHO) 33 cell line. Using immunblotting, an increase in acetylated histone H3 and H4 protein levels was observed throughout 24 hr exposure to 5 mM VPA. Secondly, to investigate whether VPA affects the activity of DNA DSB repair, CHO 33 cells were transfected with either the endonuclease I-SceI plasmid to induce a site specific DSB or the empty plasmid, pGem. However, no increase in the difference in HR between VPA and media exposed I-Sce1 transfected cells compared to cells transfected with pGem was observed, which suggests that VPA does not affect DNA repair activity. Thirdly, to determine if VPA-induced HDAC inhibition increases susceptibility to DNA damage, immunocytochemistry revealed an increase in the number of γ-H2AX foci throughout 24 hr exposure to 5 mM VPA. To determine if oxidative stress may play a role in mediating VPA-induced DNA DSBs, another recombination study was carried out in which cells were pretreated with 400 U/ml of PEG-catalase prior to VPA treatment. The observed protective effect of PEG-catalase against VPA-induced HR and the generation of intracellular ROS by VPA suggest ROS may also play a role in VPA-initiated HR. However, in our DNA oxidation study, no increase in the oxidized nucleosides, 8-hydroxy-2'-deoxyguanosine and 5-hydroxycytosine was observed after VPA treatment. These studies suggest that HDAC inhibition and ROS signalling may play other roles in DNA maintenance and cell cycle arrest in initiating DNA DSBs and HR repair. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2009-08-12 14:27:16.327

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