The eukaryotic cell cycle is governed by molecular checkpoints that ensure genomic integrity and the faithful transmission of chromosomes to daughter cells. They inhibit the cycle until conditions prevail that guarantee accurate DNA duplication and chromosome segregation. Two major mechanisms are the ‘spindle assembly checkpoint’ and the ‘DNA damage checkpoint’. During pro-metaphase, the spindle checkpoint monitors the orientation process of chromatid pairs on the bipolar microtubule array nucleated by spindle pole bodies. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, six proteins are at the heart of spindle checkpoint function: Mad1, Mad2, Mad3, Bub1, Bub3 and Mph1/Mps1. The formation of spindle checkpoint complexes signals the presence of incorrect spindle microtubule attachments to kinetochores. These complexes cooperate to suppress the activity of the anaphase promoting complex (APC) and inhibit the onset of anaphase. By isolating these distinct complexes and analysing their composition by mass-spectrometry (MS) this work revealed several intriguing disparities between the two yeast species, and the way in which the Bub and Mad proteins cooperate to achieve inhibition. The ‘mitotic checkpoint complex’, which in S.cerevisiae consists of Mad2, Mad3, Bub3 and the APC activator Cdc20, was found to lack Bub3 in S.pombe. The S.pombe complex was shown to interact with the APC, but no stable interaction was found to be required in S.cerevisiae cells. And whereas Bub1 and Bub3 were found to form a complex with Mad1 in S.cerevisiae, in S.pombe they were shown to associate with Mad3 to form the ‘BUB+ spindle checkpoint complex’. In addition, MS analysis uncovered TAPAS: a novel S.pombe complex that was found to interact with the BUB+ complex and revealed to consist of Tfg3, Abo1 (gene product of SPAC31G5.19), Pob3 and Spt16. TAPAS mutant cells were shown to lose viability as a result of genotoxic stress, a phenotype that was surprisingly shared with bub1Δ and bub1kd ‘kinase dead’ mutants. Sensitivity of cells deficient in TAPAS or Bub1 did not appear to be due to the loss of DNA damage checkpoint or DNA replication checkpoint functions. Further examination revealed that Bub1 functions in the repair of DNA double strand breaks. Taken together, this work demonstrates that even though the molecular components of the spindle checkpoint pathway are conserved, their regulatory connections have to some extent diverged through molecular evolution. This process not only rewired, but entwined two molecular processes that together safeguard the genetic heritage of cells.

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Aging-related decline in immunity is believed to be the main driver behind decreased vaccine efficacy and reduced resistance to infections in older adults. Unrepaired DNA damage is known to precipitate cellular senescence, which was hypothesized to be the underlying cause of certain age-related phenotypes. Consistent with this, some hallmarks of immune aging were more prevalent in individuals exposed to whole-body irradiation (WBI), which leaves no anatomical repository of undamaged hematopoietic cells. To decisively test whether and to what extent WBI in youth will leave a mark on the immune system as it ages, we exposed young male C57BL/ 6 mice to sublethal WBI (0.5-4 Gy), mimicking human survivor exposure during nuclear catastrophe. We followed lymphocyte homeostasis thorough the lifespan, response to vaccination, and ability to resist lethal viral challenge in the old age. None of the irradiated groups showed significant differences compared with mock-irradiated (0 Gy) animals for the parameters measured. Even the mice that received the highest dose of sublethal WBI in youth (4 Gy) exhibited equilibrated lymphocyte homeostasis, robust T-and B-cell responses to live attenuated West Nile virus (WNV) vaccine and full survival following vaccination upon lethal WNV challenge. Therefore, a single dose of nonlethal WBI in youth, resulting in widespread DNA damage and repopulation stress in hematopoietic cells, leaves no significant trace of increased immune aging in a lethal vaccine challenge model.

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, for the degree of Master of Science (Medicine). / Objective: To determine the relationship between asbestos exposure and chromosome damage, as depicted by sister chromatid exchange frequency. Design: Descriptive, cross-sectional study. Setting: Asbestos-products factory Subjects: 31 asbestos-exposed subjects and 21 unexposed subjects Main outcome measure: Mean sister chromatid exchange (SCE) frequency per metaphase cell. Results: The control group had a slightly higher mean SCE frequency per cell than the exposed group (3.4%) but this difference was not statistically significant (p = 0.5935). Smoking contributed significantly to SCE frequencies in both the exposed and unexposed groups. The mean SCE frequencies per cell in the exposed group were 10.49 for smokers and 8.59 for non-smokers (p = 0.0078). The frequencies for smokers and non-smokers in the unexposed group were 10.83 and 8.58, respectively (p = 0.0257). Conclusions: The failure to observe an increase in SCE frequency does not rule out asbestos exposure as a genotoxic agent. Rather, it may help to resolve the limitations of this method for detecting genetic damage. Alternatively, the fibre levels to which this group was exposed may have been too low to cause chromosome damage. / WHSLYP2016

by Shi Yuling. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 159-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

This study was conducted at the University of Massachusetts Medical Center in the Volkert laboratory. Human alkyl adenine DNA glycosylase (hAAG) is a DNA repair enzyme that repairs alkylated DNA bases. hAAG was cloned in 1991 and a second isoform was classified in 1994. The difference between the two isoforms of hAAG is an alternate spliced first exon. Both isoforms of the hAAG gene were present in the Volkert laboratory collection, however the second isoform (hAAG-2) was phenotypically different than the first and became the first focus of this study. Using the improperly functioning isoform as a template, and constructing a 5' primer with the identical upstream sequence as the functioning isoform (hAAG-1), a phenotypically similar gene was constructed by PCR. The new isoform (hAAG-2) was cloned into an expression vector and its activity as a DNA repair agent was studied. A second version of hAAG-2 was also constructed, incorporating a histidine tag for protein purification and identification purposes. Efforts included using the ability of hAAG to complement glycosylase deficient alkA tagA E. coli double mutant strains to assess and to compare the ability of the two isoforms of hAAG and to determine if the histidine tag affected function. The ability of hAAG to rescue cells from exposure to a variety of DNA damaging agents was studied by inducing each isoform and analyzing the sensitivity of the cells to increased doses of DNA damaging agents. Both hAAG-1 and hAAG-2 were able to restore the wild type resistance of the alkA and tag genes when exposed to the alkylating agents MNNG and MMS. In order to study the ability of hAAG to repair alkyl lesions larger than methyl groups, it was necessary to inactivate the uvrA dependent nucleotide excision repair gene. In E. coli, methyl lesions are repaired primarily by glycosylases, while nucleotide excision repairs bulky lesions. Thus, in order to detect hAAG activity on these types of damage, it was necessary to inactivate the bacterial uvrA gene. Each isoform of hAAG was transformed into a triple mutant strain deficient in alkA tagA and uvrA, then exposed to CNU, BCNU, and Mitomycin C. Each of these DNA damaging agent caused increased toxicity in the presence of hAAG. hAAG-1 expressed in the alkA tag double mutant strain was exposed to Mitomycin C and showed greater resistance than hAAG-1 expressed in the alkA tag uvrA triple mutant. In fact, in the nucleotide excision proficient strain, expression increased Mitomycin C resistance above that seen in the control, suggesting that glycosylase activity may function in a partnership with nucleotide excision repair and that the two isoforms of hAAG have subtle differences. An ompT protease knockout host strain was constructed using P1-transduction and used to examine protein products. hAAG-2 was inserted into the pBlueScript plasmid so that the gene could be regulated by the T7 promoter for use beyond the scope of this thesis. A protein synthesis time course assay was conducted to determine the expression levels of hAAG-1 and hAAG-2 when induced by IPTG. Immunoblot detection of the histidine tag was used to measure expression levels of each isoform.

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.

Constitutive activation of the ATM dependent DNA damage response and repair pathways have been reported in pre-malignant and malignant human tissues and may undermine the efficacy of genotoxic cancer therapies. Therefore, ATM inhibitors may overcome resistance to current cytotoxics and potentiate the effects of radiotherapy. A colorectal cancer model was investigated to develop a framework for the rational use of ATM inhibitors. HCT116 p21-/- cells display constitutive activation of the ATM DNA damage response but display a defect in the ionising radiation induced S-phase checkpoint, termed radioresistant DNA synthesis. This radioresistant phenotype is associated with increased basal levels of Cdc25A protein, deficient DNA damage-induced degradation of Cdc25A and Chk2 mis-localisation. HCT116 p21-/- and SW620 cells, which exhibit basal Chk2 threonine-68 phosphorylation, were unable to abrogate the S-phase checkpoint when treated with an ATM inhibitor, suggesting that the ATM– Chk2 arm is non-functional in these cells: inhibition of ATM did not potentiate the efficacy of ionising irradiation. To assess activation of the pathway a tumour microarray was created using 179 treatment naïve sporadic colorectal cancers; 152 were of the microsatellite stable phenotype. Phosphorylated Chk2 threonine-68 was present in 22 % of microsatellite-stable colorectal tumours and 33 % of tumours with the microsatellite instability phenotype. In a colorectal cancer cell line model constitutive activation of the ATM DDR pathway reflected an attenuated ATM-Chk2 axis and inhibition of ATM in these circumstances was unable to potentiate the efficacy of ionising irradiation. Basal Chk2 threonine-68 phosphorylation may reflect a deregulated ATM DNA damage response pathway and/or checkpoint adaption and therefore use of an ATM inhibitor in this background may have limited efficacy. A predictive model is proposed that integrates functionality of the ATM-Chk2 axis, p53 mutation status and defects in DNA repair pathways when considering ATM inhibitor therapy. Ultimately, molecular phenotyping and functional analysis of processes deregulated in cancer will permit individualisation of current treatment modalities, improving their efficacy and limiting patient toxicity.

CHEK1 inhibitors are currently in clinical trials for their ability to abrogate chemotherapy-induced CHEK1 activation and S phase arrest resulting in cancer cell apoptosis. No studies have yet identified ovarian cancers that could benefit from CHEK1-targeting therapy. I hypothesised that knowledge of CHEK1 and CHEK2 signalling in the DNA damage response can assist in identifying potential biomarkers for platinum responsiveness and CHEK-targeting therapy in ovarian cancer. In vitro studies investigated the CHEK1/2 inhibitor AZD7762 (AZD) and cisplatin (CP) in same patient-derived platinum-sensitive/resistant high-grade serous ovarian cancer cell lines (PEO1/PEO4 and PEO14/PEO23). Cytotoxicity assays confirmed higher CP IC50’s for PEO4 and PEO23 relative to PEO1 and PEO14 cell lines, respectively. AZD was more toxic to PEO1 cells and an additive effect of AZD with CP relative to CP alone was seen. A nontoxic AZD treatment to PEO4 cells sensitised the cells to CP when applied in combination. PEO14 and PEO23 cells had similar cytotoxicity profiles for combination treatments. BRDU DNA synthesis assays and cell cycle analysis revealed increased BRDU incorporation and accumulation in S phase when all cell lines were treated with CP. AZD treatment had a similar effect in PEO14 and PEO23 cells and increased the sub-G1 population, a marker of apoptotic DNA fragmentation, relative to control. Drug combination had no major effect on cell cycle distributions of both PEO14 and PEO23 cells relative to single agents but resulted in BRDU incorporation levels below CP and control levels for PEO14 cells. In PEO1 and PEO4 cells, AZD did not affect the cell cycle or DNA synthesis levels relative to control. Drug combination did not alter the cell cycle relative to CP treatment for PEO1 cells but decreased S phase and increased G2/M and sub-G1 populations in PEO4 cells. This was coupled with a decrease of CP-induced BRDU levels in PEO4 control levels. Apoptotic PARP cleavage/total PARP occurred early in CP treated PEO1 and PEO14 cells. A surrogate CHEK1/2 activity marker, p-CDC2 (Y15), decreased in all lines treated with AZD relative to control. Within PEO1 and PEO4 cells, greatest PARP cleavage was observed with combination treatment and coincided with high p-H2AX (S139), a DNA damage marker. p-CHEK1 (S317) and p-CHEK2 (T68), both ATR and ATM phosphorylation sites during DNA damage, increased for lone drug treatment and, to a greater extent, the combination drug treatments. PARP cleavage occurs across all treatments in PEO1 cells while it only occurs in the combination treatment for PEO4 cells. The latter coincides with a decrease in p-CHEK1 (S296) a CHEK1 autophosphorylation site, p-TP53 (S15), and p-BRCA1 (S1524), a homologous recombination marker, relative to the CP treated sample. In PEO14 and PEO23 cells, lone AZD and combination treatments had similar cleaved PARP/total PARP levels compared to the PEO14 CP treated cells. This was coupled with increased p-H2AX (S139), decreased CHEK1, and decreased CHEK2 autophosphorylation p-CHEK2 (S516). A human ovarian cancer xenograft model identified increases in p-H2AX (S139), CHEK1, p-CHEK1 (S317), p-CHEK2 (T68), and p-BRCA1 (S1524) in the carboplatin responsive cancers. In the paired pre- and post-chemotherapy human ovarian cancer samples, p-CHEK1 (S317) was elevated in post-chemotherapy responsive samples. In the first cohort, high p-CHEK1 (S317) was an independent poor overall survival biomarker and correlated with high p-H2AX (S139), MYC, p-CHEK1 (S296), p-CHEK2 (T68), p-CHEK2 (S516), and p-TP53 (S15). p-CHEK1 (S317) was associated with poor overall survival in serous ovarian cancers within the second pre-treatment ovarian cancer cohort. In conclusion, AZD can induce apoptosis in CP resistant cancer cells by synergising with CP to abrogate the S phase checkpoint, increase DNA damage, and inhibit CHEK1, and BRCA1 function. As a single agent, AZD can induce apoptosis by decreasing CHEK1 levels and CHEK2 activity. p- CHEK1 (S317) is a platinum responsive / poor prognostic biomarker.

RNF4 belongs to the family of SUMO-targeted ubiquitin E3 ligases (STUbLs). The role of STUbLs in maintaining genomic stability was first discovered in yeast. Theyeast STUbL mutants displayed genomic instability, elevated mutation rates, sensitivity to DNA damaging agents and also demonstrated synthetic lethality with other DNA repair genes. Although the role of vertebrate RNF4 in the DNA damage response was not yet established, it could rescue the Schizosaccaromyces pombe STUbL mutant phenotypes, showing that RNF4 is a functional homologue of the yeast STUbL proteins,and that it might be implicated in the vertebrate DNA damage response.A homozygous knockout of RNF4 in the DT40 chicken lymphocyte cell line was generated to investigate the involvement of vertebrate RNF4 in protecting cells against DNA damage. Although the complete loss of RNF4 did not affect cell proliferation or cell cycle distribution, the RNF4 -/- cells exhibited a selective hypersensitivity to some S-phase specific DNA damaging agents. This hypersensitivity could be rescued by introducing an ortholog of RNF4 from another vertebrate species, and this was dependent on a functional ubiquitin E3 ligase activity of RNF4.To explore the physiological function of RNF4 in the context of a wholeorganism, Danio rerio was chosen as an in vivo model. Danio rerio RNF4 sharedsimilar in vitro biochemical characteristics as RNF4 from other vertebrates – it was able to autoubiquitylate itself and also ubiquitylate SUMO2 chains. In Danio rerio, RNF4 is a maternally provided gene and is highly expressed in the adult gonads. In the ovaries, RNF4 expression was restricted to the early stage oocytes, suggesting a possible role in oocyte development. Loss-of-function studies in Danio rerio were performed using morpholino knockdown and zinc-finger knockout technologies, and the depletion of RNF4 in zebrafish did not affect early embryonic development or viability of the animal.The results presented in this thesis suggests that while vertebrate RNF4 is notlikely to be an essential gene in some vertebrates, it plays a role in the DNA damage response and might be implicated in gonad development in Danio rerio. The zinc-finger knockout model has just been established and a more in-depth analysis is necessary to shed more light on the in vivo functions of RNF4.