Translesion DNA synthesis in the context of cancer research

During cell division, replication of the genomic DNA is performed by high-fidelity DNA polymerases but these error-free enzymes can not synthesize across damaged DNA. Specialized DNA polymerases, so called DNA translesion synthesis polymerases (TLS polymerases), can replicate damaged DNA thereby avoiding replication fork breakdown and subsequent chromosomal instability

The sequence specific interaction of ionising radiation, cisplatin and T4 endonuclease VII with DNA

Cisplatin and ionising radiation are mainstays of modern cancer treatment, and both are capable of damaging DNA. They act via different mechanisms; either the formation of DNA adducts or via the induction of DNA breaks. This thesis aims to examine the genomic location of these damage sites, at nucleotide resolution, as well as examine the inherent sequence specificity of T4 endonuclease VII cleavage without the presence of a structural substrate. A similar approach was used initially – linear amplification followed by fragment analysis to determine specificity in short DNA sequences. Secondly, next-generation sequencing methods were used to detect radiation-induced damage sites in the human genome, and an attempt was made to develop a genome-wide map of cisplatin-adduct formation. The enzyme T4 endonuclease VII is a resolvase that acts on branched DNA intermediates, by cleaving DNA with staggered cuts. For the first time, the sequence specificity of cleavage sites was determined without the presence of a known DNA substrate. We found DNA was cleaved with a sequence specificity that conforms to AWTAA*STC, where A* indicates the cleavage site, W is A or T, and S is G or C. Cisplatin forms adducts with DNA, which cause distortions that prevent DNA replication. Adducts have been previously shown to form preferentially at GG nucleotides in short DNA sequences. It was shown that cisplatin adducts preferentially occurred at GG nucleotides in short DNA sequences derived from the mitochondrial genome; however, attempts to extrapolate this to a genome-wide scale were unsuccessful. Ionising radiation directly induces cytotoxic double-stranded DNA breaks, but is also subject to indirect attack from reactive oxygen species formed via radiolysis. Radiation-induced breaks are linear with respect to dose; however their precise location has not been determined. It was found that ionising radiation-induced DNA damage was sequence specific in both short DNA sequences and the entire human genome. The degree of damage at each nucleotide was quantified and it was found that the precise location of the damage site was influenced by the surrounding sequence context. This resulted in a consensus sequence of WWNGG*G in short DNA sequences and GSC*M (where M is A or C) in genomic DNA. A comparative analysis revealed an overall consensus sequence of GGSC*MC. The consistency of the consensus sequence demonstrated that damage sites are specific and non- random in nature

The interaction of platinum complexes and ultraviolet light with DNA

Two DNA damaging agents, cisplatin and UV radiation, were investigated in this thesis. For cisplatin, two studies were performed. First, the DNA unwinding angles of cisplatin and its analogues: 9AmAcPtCl2, 7-methoxy-9AmAcPtCl, 7-fluoro-9AmAcPtCl2, 9-ethanolamine-AcPtCl2 and 9AmAc were determined. It was found that all 9-aminoacridine carboxamide analogues had a smaller unwinding angle compared with cisplatin. This knowledge helps to characterize the cisplatin analogues and could facilitate in the development of a better analogue. Second, the inhibition of RecBCD exonuclease activity by cisplatin was investigated with a series of digestion assays. The enzyme was found to be inhibited by cisplatin adducts. Moreover, the inhibition was found to be dependent on the cisplatin concentration. Digestion with RecBCD enabled a reduction of DNA length until a cisplatin adduct was reached. This suggested that RecBCD could be employed to reduce the large number of DNA sequences and enrich adduct-containing DNA sequences for Next-Generation sequencing. Another focus of this thesis was the investigation of the interaction between UV radiation and DNA. Two studies were performed to build knowledge on the topic. First, we employed the linear amplification technique and the end-labelling approach followed by capillary electrophoresis with laser-induced fluorescence to investigate the hexanucleotide consensus sequence for UV-induced DNA damage in two sequences that were specifically designed for UV damage analysis (the TanUV and X-ray clone plasmid). Two consensus sequences were derived from the end-labelling analysis that mainly detects 6-4PPs, 5'-GTTC*CC and 5'-ACCC*GT. The results also showed (in a different DNA sequence) that at the first and the last position of a hexanucleotide, an A nucleotide variant increased the level of 6-4PP damage significantly. Second, the effect of methylation on the level of UV damage to DNA was investigated. A purified isolated DNA with known sequence (CG plasmid) was methylated with CpG methylase. The levels of CPD and 6-4PP adducts detected in methylated/unmethylated labelled sequences were analysed. The comparison showed that 5-methyl-cytosine reduced the level of both CPD and 6-4PP significantly after UVB (308 nm) and UVC (254 nm) irradiation compared with the non-methylated counterpart. Moreover, we demonstrated that for 6-4PP adducts, a higher dosage of UVB and UVC irradiation further reduced the methylated/non-methylated ratio

Investigating enzyme communication during base excision repair in Escherichia coli

Mismatch uracil DNA Glycosylase (MUG) from Escherichia coli is an initiating enzyme in the base excision repair (BER) pathway and is responsible for the removal of 3,N4-ethenocytosine and uracil from DNA during the stationary phase of E.coli cell growth. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. MUG is widely regarded as a “single turnover” enzyme because it still remains tightly bound to its abasic product after cleavage, thus impeding its catalytic turnover. This may be a general protective mechanism to protect the abasic BER intermediate, whereby coordination of enzyme activity in BER is achieved through displacement of the DNA glycosylase by the downstream apurinic-apyrimidinic (AP) endonuclease. Numerous DNA glycosylases have now been cited as having an enhanced turnover in the presence of an AP endonuclease. The aim of this project is to investigate enzyme coordination between MUG and its both downstream AP endonucleases, Exonuclease III (ExoIII) and Endonuclease IV (EndoIV), in the initial steps of BER. We show here that MUG binds its substrate, abasic DNA and non-specific DNA in the differential modes. A 2:1 cooperative binding stoichiometry with abasic DNA is demonstrated to be of functional significance in both product binding and catalysis via fluorescence anisotropy assays, band shift assays and loss-of-function site-directed mutagenesis methods. The effects of the ExoIII and EndoIV on the MUG turnover kinetics with a U•G containing substrate was investigated. Both ExoIII and EndoIV greatly enhance the turnover of MUG. Furthermore, the analysis of both ExoIII catalytic activity dependent and concentration dependent on MUG turnover demonstrate ExoIII may employ a product scavenging mechanism to enhance MUG turnover. These combined results constitute a new concept that MUG has a pre-catalytic discrimination ability to coordinate its reactivity behavior with the other enzymes.Open Acces

Spontaneous mutagenesis in stressed <i>Escherichia coli</i>

Mutation processes occurring in starving or stressed bacteria are different from those that are found in growing bacteria. The mutageitic response of E.coli to a number of different stress conditions, including amino acid starvation and challenge with the antibiotic streptomycin has been examined. In an E.coli trpA23 strain starved for tryptophan, the formation of small in-frame deletions was stimulated by the persistence of oxidatively damaged guanine and unrepaired mismatches in the DNA. Most (75%) of the deletions had direct repeats at each termini, one of which was lost in the deletion process. The deletions restored functionality to the product of the trpA gene enabling the mutants to form slow growing colonies on minimal plates lacking tryptophan. During starvation of an E.coli tyrA14 strain WU3610 for the amino acid tyrosine, a new phenotypic suppressor gene (tas) was isolated that specifically complemented the absence of prephenate dehydrogenase activity. The gene is necessary for the process of starvation-associated mutagenesis in this strain; mutants do not arise during starvation in a ray deletion strain. A high proportion (up to 20%) of ancillary mutations within the rpsL gene of E.coli have previously been found in newly arising streptomycin dependent mutants and have been shown to confer a selective advantage in established strains. This study shows that they also confer an advantage in mutants containing mixed wild type and streptomycin dependent ribosomes in the presence and absence of streptomycin. However, this selective advantage is still not enough to account for the rate at which these ancillary mutations are recovered and we need to assume a mutation rate of 10-3 to 10-4/gene/replication to explain the observed frequency

Molecular analysis of DNA damage induced by a novel trinuclear platinum complex (BBR 3464)

We evaluated the activity of the trinuclear platinum complex BBR 3464 in two human ovarian carcinoma cell lines (OAW42, A2780) and in their cisplatin-resistant counterparts (OAW42Mer, A2780cp8). An increased cytotoxic potency of BBR 3464 compared to cisplatin was generally observed, an a collateral sensitivity or a very modest cross-resistance to BBR 3464 was found in OAW42Mer and A2780cp8 cell lines, respectively. Loss of mismatch repair proteins (hMLHl, hPMS2) or overexpression of nucleotide excision repair proteins (ERCC1) was not detrimental for the cellular sensitivity to the trinuclear platinum complex. BBR 3464 intracellular accumulation and DNA-bound platinum were consistently higher than those observed with cisplatin. After exposure to BBR 3464 and cisplatin of purified DNA or intact cells, a similar sequence preference of DNA damage was observed. Conversely, interesting differences in the kinetics of formation and removal of DNA lesions at the single-gene (N-ras) level were observed between the two drugs. The interference exerted by BBR 3464 with cell cycle progression and its ability to induce apoptosis were evaluated in OAW42 and OAW42Mer cell lines. Flow cytometric experiments indicated that in the two cell lines BBR 3464 was able to induce a persistent G2M block whereas cisplatin caused an initial accumulation of cells in the S phase followed by an increase in the G2M cell fraction. Exposure to IC50 drug concentrations induced apoptosis in both cell lines. However, the percentage of cells with an apoptotic nuclear morphology was slightly higher after cisplatin than BBR 3464 treatment in OAW42 cells, whereas the opposite pattern was observed in OAW42Mer cells. Degradation of the nuclear lamin B was detected in OAW42 cells after exposure to each drug whereas in OAW42Mer cells the cleavage was only appreciable after BBR 3464 exposure. In OAW42 cells the mitochondrial membrane potential (ɅѰmt) was not affected by the two drugs, whereas in the OAW42Mer cell line a marked (ɅѰmt) reduction was observed only after exposure to BBR 3464. Overall, the results would suggest that the collateral sensitivity to BBR 3464 observed in the OAW42Mer cell line might be attributable to the ability of this drug to modify DNA differently from that of cisplatin and, as a consequence, to induce different cellular responses to DNA damage such as the triggering of specific apoptotic pathways

The effect of metronidazole on Bacteroides fragilis and Escherichia coli

The antibiotic metronidazole is used extensively in the clinical treatment of anaerobic infections, including those caused by the anaerobic pathogen Bacteroides fragilis. Metronidazole is an inert substance that requires reductive activation to become cytotoxic. In its activated form metronidazole induces DNA damage. Relatively little is known about the cytotoxic effects of this drug in vivo. The aim of the work reported in this thesis was to analyze the mode of action of metronidazole in living systems. Furthermore, the potential for bacterial cells to develop resistance mechanisms to metronidazole is largely unknown, and therefore the role played by B. fragilis genes in influencing the potency of metronidazole was investigated. Bibliography: pages 172-201