No description available.

Unrepaired, or misrepaired, DNA damage can be carcinogenic or mutagenic; thus functional DNA damage repair pathways are essential for the safeguarding of the genome. SNM1B is a 5' to 3' exonuclease implicated in the repair of damaged DNA, particularly the repair of interstrand crosslinks. Genetic studies have identified SNPs in the SNM1B gene as related to cancer risk. One of these (rs11552449) encodes a single amino acid change, H61Y. This study shows that WT and H61Y SNM1B have comparable in vitro biochemical and biophysical characteristics. The structures of both WT and H61Y C-terminally truncated SNM1B (Δ-SNM1B) were solved to 2.8 and 3.1 Å, respectively, and reveal similar structural properties. The structure of WT Δ-SNM1B was also reported (to 1.8 Å) with two 2'-deoxy-5'- adenosine monophosphate molecules in the active site. The structure of SNM1B shows an accessible extended active site, which may facilitate the binding of a variety of non-canonical DNA substrates. Accordingly, in vitro, WT and H61Y SNM1B are able to exonucleolytically process a wide range of structurally diverse DNA substrates. By utilising SNM1B depleted cell lines, this study also shows that SNM1B is required for DNA repair in response to treatment with DNA-crosslinking genotoxic agents (including cisplatin and SJG-136). This study also identifies the novel double strand break repair factor, EXD2, as having intrinsic 3' to 5' exonuclease activity. EXD2 was shown to have enzymatic activity on a variety of substrates in vitro, including replication fork intermediates, 'nicked' or 'gapped' DNA duplexes, and RNA based substrates. Together with the cellular data this suggests a role for EXD2 in nucleolytically processing RNA or DNA-based intermediates in damage repair pathways.

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.

No description available.

CRISPR/Cas9 is the gene editing tool of choice in basic research and poised to become one in clinical context. However, current studies on the topic suffer from a number of shortcomings. Mutagenesis is often assessed using bulk methods, which means rare events go undetected, unresolved or are discarded as potential sequencing errors. Many of the genotyping methods rely on short-range PCR, which excludes larger structural variants. Other methods, such as FISH, do not provide basepair resolution, making the genotype assessment imprecise. Furthermore, it is not well understood how Cas9 delivery format influences the dynamics of indel introduction. Finally, many studies of on-target activity were conducted in cancerous cell lines, which do not accurately model the mutagenesis of normal cells in the therapeutic context. In my thesis, I have investigated on-target lesions induced by Cas9 complexed with single gRNAs and no exogenous template. I have followed the time dynamics of Cas9-induced small indels as a function of reagent delivery methods, established an assay for quantification of Cas9-induced genomic lesions that are not small indels ("complex lesions") and used this assay to isolate and genotype complex lesions, many of which would be missed by standard methods. I found that DNA breaks introduced by single guide RNAs frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and cross-over events were identified. Frequent and extensive DNA damage in mitotically active cells caused by CRISPR/Cas9 editing may have pathogenic consequences.

No description available.

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)

Chromodomain Helicase DNA-Binding Protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double stranded break (DSB) repair. Here, we show that depletion of CHD4 in Acute Myeloid Leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ATM pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic-agent induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34+ hematopoetic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor formatting behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.

DNA damage repair is an essential and complex cellular process. Although the basic mechanisms of nucleotide excision repair (NER) have been studied for decades, some mechanistic details remain elusive. The lesion detection step remains one of the most elusive in the process of NER in the contest of chromatin. The work described herein addresses the initial events in the lesion detection step of chromatin repair, also referred to as global genome repair (GG-NER). Both the role of post-translational modifications of lesion identification proteins, and the initial sequence of events in recruitment of repair and remodeling factors are investigated. First, the controversial role of ubiquitination of DDB2 (a human lesion detection protein) is investigated. Due to documented DDB2 function in alternative physiological processes, its direct role in GG-NER is hard to study in human cells. To overcome this obstacle, we established the budding yeast, Saccharomyces cerevisiae as an alternative, simplified model organism to study DDB2-mediated GG-NER. Using this system, we show that inconsistent with the widely accepted model, rapid degradation of DDB2 post-UV irradiation is not an absolute requirement for progression of GG-NER. However, interestingly, our data suggest a role for ubiquitination in the release of DDB2 from chromatin. In both UV and mock treated samples, ubiquitin deficient cells had significantly higher amounts of DDB2 remaining bound to the chromatin compared to the isogenic parent cells. The discussion focuses on the possible physiological relevance of these observations. Additionally, the recruitment of the SWI/SNF chromatin remodeling complex to the silent HML (Hidden MAT Left) locus was also investigated. SWI/SNF is known to require recruitment for its role in transcription; therefore we investigate this requirement in GG-NER. Based on previously published data that indicate an UV-stimulated association of SWI/SNF and Rad4 (a lesion detection protein), we hypothesized that Rad4 is involved in recruitment of SWI/SNF to damaged DNA. Interestingly, our data suggest that Rad4 is not an absolute requirement for recruitment of Snf6 to the HML locus following UV irradiation. However, Rad16 appears to be. These data present an interesting insight into the lesion detection step in GG-NER and this will be discussed.

Ewing Sarcoma (ES) is an aggressive pediatric solid tumor. Even though overall-survival for localized patients is approximately 70%, the overall-survival for high risk ES patients has not improved in the last 20 years. Therefore, there is a need for exploration of new therapeutic agents in ES. Recent evidence has demonstrated that ES cells behave like BRCA-deficient tumor types which renders them sensitive to PARP inhibitors in vitro and in vivo. However, a phase II study of the efficacy of single-agent PARP inhibition in patients with relapsed ES did not significantly improve outcome. As single-agent therapy is rarely expected to result in significant clinical responses, in this study, we plan to validate potential targeted combination therapies with PARP inhibitors in ES. Since ES appears to demonstrated BRCA-deficient biology with impaired homologous recombination, cells are expected to be sensitive to both PARP inhibitors and ATR inhibitors, drugs which have a role in regulating DNA damage and impairing homologous recombination. In breast cancer and ovarian cell lines with genetic BRCA-deficiency, PARP and ATR inhibitors have synergistic activity. We hypothesize that these inhibitors will also have synergistic anti-Ewing activity. Furthermore, we recognize that ES cells demonstrate remarkably quiet genomes suggesting that there is minimal ongoing DNA-damage when cells are growing unperturbed. Therefore, we also plan to test the effect of adding low-dose genotoxic chemotherapy to induce additional sensitivity to the combination of PARP and ATR inhibitors in ES. The specific aims of this study were to explore the possible anti-tumor effect of PARP inhibitors combined with ATR inhibitors in ES cell lines, and to explore whether low dose genotoxic chemotherapy with SN38 can potentiate the anti-tumor effect of combined PARP and ATR inhibition in ES cell lines. We studied the anti-Ewing Sarcoma effect of the combination of a PARP inhibitor, AZD2281, and an ATR inhibitor, AZD6738, across a range of doses with and without low doses of a DNA damaging agent, SN38 (irinotecan metabolite), in two ES cell lines. We analyzed synergy by determining the Combination Index (CI) and Fractional Inhibition (FA) of each combination. We found that the ATR inhibitor, AZD6738, was synergistic across large range of concentrations when combined with the PARP inhibitor, AZD2281, in ES cell lines. We also found that treatment of cells with low doses of SN38 increases ES cell sensitivity to treatment with the PARP inhibitor and ATR inhibitor combination. This study provides preclinical support for additional studies exploring these combinations in ES. Given the low number of pediatric patients with ES compared to adult cancer patients, there will be limited attempts in combining these agents in clinical trials. Therefore, the development of an in vivo trial testing the safety and efficacy of this combination in ES mouse models is proposed. / 2020-07-03T00:00:00Z