The base excision repair protein, MBD4 (also known as MED1) is known to be transcriptionally coupled to a mismatch repair protein MLH1. To date the significance of this coupling has not been elucidated and the significance of MBD4 within the mismatch repair system and apoptotic pathway is still being understood. Recently a novel alternatively spliced form of MBD4 has been identified and termed sMBD4. To date the significance of sMBD4 is unknown. MBD4 and sMBD4 share a common glycosylase domain and this is the domain through which MBD4 is reported to interact with MLH1. It was the aim of this study to determine if sMBD4 was also a binding partner of MLH1 to help elucidate a potential role of sMBD4 and to further characterise the binding domain between MLH1 and MBD4. Recombinant proteins were utilised in binding assays however, a specific protein – protein interaction could not be determined. Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of DNA mismatch repair (MMR) proteins in DNA MMR proficient cells. As MBD4 has previously been suggested to be coupled to MLH1 expression by a post‐translational mechanism the cytotoxicy of aspirin in relation to MBD4 expression was examined. This study reports that aspirin does not up‐regulate MBD4 gene transcription in vitro in the DNA mismatch repair proficient/p53 mutant colorectal cancer cell line SW480. However, MBD4 gene transcription was up‐regulated upon treatment with the aspirin precursor, salicylic acid. The suggested involvement of the DNA repair proteins in the mechanism of action of aspirin promoted the investigation into the expression of DNA damage signalling pathways genes upon aspirin exposure. This study utilised a commercially available PCR array to analyse the expression of 84 DNA damage signalling genes in the SW480 colorectal cancer cell line upon aspirin treatment. It is reported that treatment of the SW480 cell line with aspirin caused changes in mRNA expression of several key genes involved in DNA damage signalling including a significant down‐regulation in expression of the genes encoding ATR, BRCA1 and MAPK12 and increases in the expression of XRCC3 and GADD45α genes. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo‐protective effect of aspirin in vivo.Further to this, protein expression was analysed to determine if correlation could be established with the changes in mRNA expression observed. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45α, an increase in XRCC3 protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This study indicates that alterations in gene expression seen in microarray studies need to be verified at the protein level. Furthermore, this study reports the novel discovery of XRCC3 gene and protein expression being susceptible to exposure to the non‐steroidal anti‐inflammatory drug, aspirin.

Generation and phenotypic analyses of stzA gene deletion strains of Aspergillus nidulans implies that stzA is allelic to sltA, with the encoded transcription factor regulating tolerance to cations, DNA-damaging agents and high arginine concentrations. The similar severe sensitivity of a sltA1 mutant (GO281) and stzA deletion mutants to these stresses indicated that the premature termination codon in sltA1 represents a total loss-of-function mutation. It was also verified that StzA has no regulatory role in the utilisation of carbon sources. Findings were supported by phenotypic analyses of recombinant progeny resulting from sexual crosses between sltA1 and sltA+ strains. Bioinformatic analysis of genes involved in the osmotic stress response revealed that their promoters were significantly enriched for StzA binding site motifs compared to those of the control group, indicating that StzA may regulate many of these genes that comprise the High Osmolarity Glycerol (HOG) pathway. Although this pathway is activated by fludioxonil, stzA deletants and stzA+ strains showed similar sensitivities to this fungicide. Phenotypic analyses indicate that StzA does not regulate tolerance to sources of oxidative stress, non-ionic osmotic stress or components of the Cell Wall Integrity (CWI) pathway. A. nidulans StzA appears to have no role in cellulase or xylanase expression as revealed by the results of a dinitrosalicylic acid (DNS) assay. Trichoderma reesei ace1 deletant and wild-type strains showed similar sensitivities to cations, DNA-damaging agents, arginine, neomycin, acidic and alkaline pH. These results confirm that A. nidulans StzA and T. reesei Ace1 regulate the distinct phenotypes of abiotic stress tolerance and cellulase and xylanase expression, respectively, despite these two proteins sharing 58% overall amino acid similarity. All twenty-nine StzA orthologues identified are restricted to filamentous ascomycetes of the Pezizomycotina subphylum and may therefore represent specific and novel antifungal drug targets. The C-termini of StzA proteins are highly variable in both length and sequence, with no apparent conservations in amino acids or predicted secondary structure. This region is considered most likely to influence the divergent functions of StzA proteins. Conservations of individual residues in the N-termini correspond to conserved secondary structure (alpha helices) among StzA proteins, implying shared functions for StzA proteins in this region. Regulators of two major nitrogen metabolic pathways (CpcA and AreA) may regulate stzA expression. Statistically significant putative CpcA binding sites were positionally conserved in 26 out of 29 stzA orthologue promoters, indicating an interaction between stzA and CpcA, a transcription factor that mediates the cross pathway control of amino acid biosynthesis. REALALE sequences, likely to be of retrotransposon origin, containing putative overlapping binding sites for StzA and AreA, were found in eleven stzA promoters of the Eurotiomycetes class, indicating an interaction between stzA and the global nitrogen metabolite repressor AreA. Intriguingly, REALALE-containing promoters identified across the genome of A. nidulans were significantly enriched for StzA binding site motifs when compared to a control group of genes. Hence, REALALE may have regulatory significance that extends to other A. nidulans genes.

No description available.

Sources of arsenic exposure include air, water, and food from both natural and anthropogenic sources. Arsenic is categorized as a human carcinogen, and is associated with pleiotropic toxicities including cancers of the skin, lung, and bladder. Despite arsenic's long recognition as a human carcinogen, the exact mechanisms of arsenical-induced carcinogenesis are unknown. Arsenic exposure has been shown to cause DNA damage. However, because arsenic does not directly react with DNA, genotoxicity is generally considered to result from indirect mechanisms. The generation of arsenical-induced reactive oxygen species and the inhibition of critical DNA repair systems are believed to contribute to arsenical-induced carcinogenicity. The DNA damaging effects of arsenical exposure and alterations in DNA repair processes were examined within the human bladder urothelial cell line, UROtsa, following continuous exposure to the arsenic metabolite, monomethylarsonous acid [MMA(III)]. Chronic, low-level MMA(III) exposure results in the induction of DNA damage that remains elevated following the removal of MMA(III). Furthermore, data presented herein, defines the critical period in which continuous low-level MMA(III) exposure causes the malignant transformation of the UROtsa cell line. Results indicate that malignant transformation of UROtsa cells is irreversible following 12 wk of low-level MMA(III) exposure. Assessment of the MMA(III)-induced biological alterations leading to the malignant transformation of UROtsa cells following 12 wk of exposure suggest two potential interdependent mechanisms in which MMA(III) may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation. These mechanisms include MMA(III)-induced DNA damage via the production of reactive oxygen species and the MMA(III)-induced inhibition of poly(ADP-ribose) polymerase-1 as a result of the direct MMA(III)-mediated displacement of zinc.

Our cells are constantly dealing with DNA damage generated by endogenous cellular activity (e.g. DNA replication) and exogenous agents (e.g. ultraviolet and ionizing radiation (IR)). The cellular stress response to DNA damage requires strict co-ordination between cell cycle checkpoint control and DNA repair. In response to DNA double-strand breaks (DNA-dsbs), members of the phosphatidylinositol 3-kinase–related kinase family (e.g. ATM and DNA-PKcs kinases) have been shown to redundantly phosphorylate substrates including the DNA-dsb marker, gamma-H2AX, and the p53 tumour suppressor protein. The p53 protein is best known as the guardian of the genome through its transcriptional-dependent and -independent functions. Despite a clear link between ATM-dependent phosphorylation of p53 with cell cycle checkpoint control and various modes of DNA damage repair, the intracellular biology and sub-cellular localization of p53 and specifically its phosphoforms during DNA damage induction and repair remains poorly characterized. Using G0/G1 confluent primary human diploid fibroblast cultures, this thesis shows that endogenous p53, phosphorylated at serine 15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following the induction of DNA breaks. This biologically distinct sub-pool of p53Ser15 is ATM-dependent and resistant to 26S-proteasomal degradation. p53Ser15 co-localizes and co-immunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA-dsb rejoining. Sub-nuclear microbeam irradiation studies confirm that p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATMSer1981 and DNA-PKcsThr2609 in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX-association and altered DNA-dsb kinetics following DNA damage. We further hypothesized that the non-specific DNA binding activity of the p53 carboxy-terminus mediates chromatin anchoring at sites of DNA damage. YFP-p53 fusion constructs expressing carboxy-terminus deletion mutants of p53 were transfected into p53-null H1299 cells to determine the role of the carboxy-terminus in chromatin-binding pre- and post-IR, independent of transcriptional activity. Within this isogenic human cell system, we observed exogenous YFP-p53WT associated with ATMSer1981 and 53BP1 within cellular chromatin in a dynamic manner. We confirmed that these associations also occurred between endogenous WTp53 with ATMSer1981 and 53BP1 within the chromatin of primary human diploid fibroblasts. YFP-p53del1-299 fusion proteins, which lack transcriptional activity and the Ser15-residue, also associated within chromatin. Ser15-phosphorylation was found not to be essential for DNA damage-induced association of p53 with chromatin or with ATMSer1981 and 53BP1. These data suggest a unique biology for p53Ser15 phosphoforms in the initial steps of DNA damage signaling and implicates ATM-p53-53BP1 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair. And we propose a model whereby a pre-existing pool of p53 that constantly scans the genome, responds immediately to radiation-induced DNA damage by virtue of its association with chromatin through its carboxy-terminus. The consequences for these p53-ATMSer1981-53BP1 complexes following DNA damage remains to be investigated: could residual complexes be associated with decreased DNA-dsb rejoining or error-prone repair, or could these complexes signal for cell survival or cell death? Since altered p53 function and biology is an important factor in cellular carcinogenesis and response to cancer therapy, this study provides a step towards a greater understanding of WTp53 and MTp53 biology in tumour development and therapeutic resistance, in the hopes to contribute towards predicting therapeutic response and/or improving p53-targeted therapies.

Caspase-3 has demonstrated a non-apoptotic function in several developmental programs including skeletal muscle differentiation, yet the mechanism of action has not been fully elucidated. Under apoptotic conditions Caspase-3 induces DNA fragmentation through activation of CAD. Recent observations have demonstrated CAD activity and the resulting DNA strand breaks are also vital for skeletal muscle differentiation. These breaks are transient in nature, suggesting an active DNA repair program to maintain genomic integrity. The aim of this study was to delineate the DNA repair mechanism coordinated with caspase/CAD mediated DNA damage. It was found that XRCC1 formed punctate nuclear foci early in myoblast differentiation concurrent to the induction of DNA damage. Caspase-3 inhibition caused attenuation of the formation of DNA lesions and XRCC1 foci in differentiating myoblasts. Targeted reduction in XRCC1 expression impaired myoblast differentiation. These results suggest that XRCC1 may play a role in repairing the DNA damage associated with myoblast differentiation.

Petrol attendants are exposed to petrol volatile organic compounds (VOCs) which may have genotoxic and carcinogenic effects. The single cell gel electrophoresis assay (comet assay) is a method highly sensitive to DNA damage induced by environmental and occupational exposure to carcinogenic and mutagenic agents. The aim of this study was to evaluate the level of exposure of petrol attendants to petrol VOCs and also to determine their effect on DNA damage and repair in lymphocytes of African petrol attendants. The exposed group consisted of 20 subjects, randomly selected from three petrol stations. A control group of 20 unexposed subjects was also chosen and matched for age and smoking habits with the exposed group. Sorbent tubes were used to assess personal exposure of petrol attendants. The comet assay was used to investigate the basal DNA damage and repair capacity in isolated lymphocytes of petrol attendants and control subjects. Blood samples were taken from the petrol attendants at the end of their 8 hour working shift and also from the control subjects. The petrol attendants were found to be exposed to levels of petrol VOCs lower than the occupational exposure limit (OEL) for constituent chemicals. A significant relationship was found between the volume of petrol sold during the shift and the average concentrations of benzene, toluene and the total VOCs measured. However, relative humidity had a negative correlation with the average concentrations of benzene, toluene, xylene and the total VOCs. Significantly higher basal DNA damage was observed with the exposed group compared to the control group. The period of exposure influenced the level of DNA damage and the calculated repair capacity. Smoking and age had a significant influence on the level of DNA damage. DNA repair capacity was delayed in smokers of both exposed and non-exposed group. / Thesis (M.Sc. (Occupational Hygiene))--North-West University, Potchefstroom Campus, 2008.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine degradation pathway. The defective fumarylacetoacetate hydrolase enzyme causes the accumulation of upstream metabolites such as fumarylacetoacetate (FAA), maleylacetoacetate (MAA), succinylacetone (SA) and p-hydroxyphenylpyruvic acid (pHPPA). In vitro and in vivo studies showed that the accumulation of these metabolites are detrimental to cell homeostasis, by inducing cell cycle arrest, apoptosis, and endoplasmic reticulum stress, depleting GSH, inhibiting DNA ligase, causing chromosomal instability, etc. For in vivo studies different models of HT1 were developed. Most notably was the fah deficient mouse, whose neonatally lethal phenotype is rescued by the administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). Although, this model most closely resembles the human phenotype with elevated tyrosine levels and the development of hepatocellular carcinoma (HCC), the model is not human genome based. Both the in vitro and in vivo studies suggested that DNA repair is affected in HT1. However, it is not yet clear which DNA repair mechanisms are affected and if only protein functionality is affected, or if expression of DNA repair proteins are also affected. Characteristic of HT1 is the high prevalence of HCC and the presence of liver mosaicism. The liver mosaicism observed in HT1 patients are the result of reversion of the inherited mutation to wild-type. The general consensus is that the reversion is the result of a true back mutation. However, the mechanism underlying the back mutation is still unresolved. It was suggested that cancer develops either through a chromosomal instability mutator phenotype, a microsatellite instability mutator phenotype, or a point mutation instability mutator phenotype. In HT1 only chromosomal instability was reported. The aims of this study were to contribute to the understanding of the molecular basis of the genetic mosaicism in hereditary tyrosinemia type 1. More specifically, determine whether baseand nucleotide DNA repair mechanisms are affected and to what extent, and to determine if microsatellite instability is found in HT1. To achieve these aims, a parallel approach was followed: i.e. to develop a HT1 hepatic cell model and to use HT1 related models and HT1 patient material. To assess the molecular basis of the genetic mosaicism in HT1, the comet assay, gene expression assays, microsatellite instability assays, high resolution melting and dideoxy sequencing techniques were employed. Results from the comet assay showed that the HT1 accumulating metabolites, SA and pHPPA, decreased the capacity of cells for base- and nucleotide excision repair. Gene expression assays showed that short term exposure to SA and/or pHPPA do not affect expression of hOGG1 or ERCC1. The expression of these genes were, however, low in HT1 patient samples. Microsatellite instability assays showed allelic imbalance on chromosome 7 of the mouse genome, and microsatellite instability in the lymphocytes of HT1 patients. Although high resolution melt and sequencing results did not reveal any de novo mutations in fah or hprt1, the appearance of de novo mutations on other parts of the genome can not be ruled out. To conclude, results presented in this thesis, for the first time show that in HT1 the initiating proteins of the base- and nucleotide repair mechanisms are affected, the gene expression of DNA repair proteins are low, and microsatellite instability is found in HT1. By contributing to the elucidation of the mechanism underlying the development of HT1-associated HCC, and providing evidence for the development of a mutator phenotype, the results presented in this thesis contributes to the understanding of the molecular mechanisms underlying the genetic mosaicism in HT1. In addition to these contributions, a hypothesis is posited, which suggests that a point mutation instability (PIN) mutator phenotype is the mechanism underlying the mutation reversions seen in HT1. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Defective DNA repair capacity has been shown to be a common feature of cancer, and loss of function mutations in 'stability' genes that normally maintain the integrity of the genome may prove a key rate-limiting step in carcinogenesis. Since even genetically unstable cells require some repair functionality to maintain viability, these cancers likely exhibit an over-reliance on other DNA repair pathways for survival. Therapeutically targeting backup repair processes in such tumours represents a novel means by which to achieve selective tumour toxicity. Full exploitation of these synthetic lethal interactions will require an in-depth knowledge of the genetic basis of DNA repair in combination with an armoury of small molecule inhibitors of cellular targets. To this end, we have designed, optimised and run two high-throughput cell-based screens to identify genes and small molecules that can modulate mismatch repair (MMR) activity. Key to these screening strategies are the resistance of cells with dysfunctional MMR to a range of cytotoxic drugs, including the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). By exploiting this MMR-dependent toxicity we have assayed for siRNA and small molecules that permit the survival of MNNG-treated MMR-proficient cells to levels comparable to MMR-deficient cells, and which therefore represent putative MMR modulating agents. A screen of 571 siRNA for gene depletions that reduce MNNG sensitivity by at least two population standard deviations identified 10 genes of potential interest, and included the four canonical MMR genes, MSH2 (2.87 ± 0.28 (Z ± SE)), MSH6 (4.87 ± 0.06), MLH1 (3.42 ± 0.43) and PMS2 (3.36 ± 0.44). TDG represented an unexpected hit that decreased MNNG sensitivity by 2.55 ± 0.04 population standard deviations. However, clonogenic survival experiments found TDG depletion to be contextual synthetic lethal within an MMR-null background when treated with MNNG, reducing HCT116 clonogenicity by 37% (p < 0.001). Moreover, TDG knockdown increased the number of 53 binding protein 1 (53bp1) foci in MMR-proficient cells by 40% and MMR–deficient cells by 27% following MNNG exposure (p < 0.001). Combined with a failure to replicate the primary screen result, the role for TDG in the response to MNNG could be explained solely through its established role as a member of the base excision repair pathway. A second screen of the NCI Diversity I and II small molecule libraries (n=1786) was conducted to identify putative MMR inhibitors. Subsequent analysis revealed NSC197049 to increase cellular viability of MNNG treated cells by 3.60±0.32 population standard deviations and was successfully validated as a hit. Co-treatment of NSC197049 with MNNG conferred dose-dependent chemoprotection independently of MMR status and cell line, an effect that was lost if NSC197049 was pre- or post-treated. The protection was associated with a reduction in MNNG-dependent 53bp1 foci of 60% in MMR proficient cells and 15% in MMR deficient cells (p < 0.001), together with a marked reduction of > 80% in subG1 content at 48 hours post-MNNG that was independent of MMR status. Interestingly, the characteristic G2/M arrest of MNNG-treated MMR-proficient cells remained intact (~40% arrested). Taken together, these observations are not consistent with NSC197049 acting as an inhibitor of MMR. Dithiolthiones have been described as chemoprotective agents that induce antioxidant defences, whilst we have found NSC197049 phenocopies known antioxidants ascorbic acid and glutathione in protecting against MNNG-induced toxicity. NSC197049 may therefore act by bolstering cellular antioxidant defences. The precise mechanism may be novel, since the proto-typical dithiolthione, Oltipraz, failed to be protective in this study. In summary, we have confirmed that MMR is the primary determinant of MNNG sensitivity, and found that TDG is unlikely to be involved in MMR. We have also identified a novel chemoprotective small molecule that is unlikely to represent an MMR inhibitor, but that might be useful in cancer chemoprevention.

Cdt1 is a conserved and essential eukaryotic protein that is required for the licensing step of DNA replication. In order to control replication licensing and ensure a single round of DNA replication occurs per cell cycle, Cdt1 is subject to strict regulation. In Metazoa and S. pombe, Cdt1 is targeted for ubiquitylation and proteolysis in S phase and after DNA damage by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 is activated in Metazoa by an unusual mechanism that requires an interaction between the substrate and chromatin-loaded proliferating cell nuclear antigen (PCNA). This study addressed the involvement of PCNA in S. pombe Cdt1 proteolysis. A mutational analysis was undertaken to establish whether the Cdt1-PCNA interaction is conserved in S. pombe and the extent to which it regulates CRL4Cdt2-dependent turnover of the protein. S. pombe Cdt1 was shown to interact with PCNA in vivo and two variant PCNA-interacting peptide (PIP) motifs were identified in the protein. The two motifs function near-redundantly to promote both the Cdt1-PCNA interaction and the CRL4Cdt2-dependent proteolysis of Cdt1 in S phase and after DNA damage. The mutational analysis also resulted in the characterisation of two in-frame AUG codons in the cdt1+ reading frame. The second in-frame AUG codon was shown to be the principal initiator codon and was required to maintain wildtype Cdt1 protein levels and cell viability. CRL4Cdt2 is emerging as an important regulator of proteins that are involved in the control of cell cycle progression and the maintenance of genome stability. However, there are a number of outstanding questions regarding the mechanism and regulation of CRL4Cdt2. In order to address these questions, a genomics approach was taken to identify novel genes involved in Cdt1 regulation. A screen of non-essential S. pombe genes identified 17 candidate genes that, when inactivated, caused up-regulation of Cdt1. Unexpectedly, deletion of genes involved in homologous recombination resulted in a Rad3-dependent up-regulation of Cdt1. Further work is required to establish the biological significance of this finding.