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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 se

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 hu

Cisplatin is a highly successful DNA-damaging antitumour drug that has been employed for decades in the treatment of ovarian, testicular, lung, and other cancers. However, it faces a number of shortcomings including drug resistance and dose-limiting toxic side effects. A number of cisplatin analogues have been developed to overcome these issues, such as carboplatin and oxaliplatin. This project aimed to characterise the DNA binding properties of several 9-aminoacridine linked platinum analogues that show good potential in antitumour efficacy. These analogues contain an intercalating group that alters the sequence specificity of DNA binding and also increases DNA binding efficiency.Methylation of DNA occurs at CpG motifs and is frequently aberrant in the promoter regions of tumour cells. Given that the 9-aminoacridines have a preference to bind to CpG motifs, we were interested in determining what effect methylation has on the DNA binding of these 9-aminoacridine linked platinum analogues. These studies found that methylated CpG increases the binding frequency of the 9-aminoacridine analogues to DNA, while the binding of cisplatin was unaffected. This is of interest as an increased efficiency of DNA binding by these analogues to methylated DNA could result in better efficacy of treatment in tumour cells.An ethidium bromide displacement assay was performed on the 9-aminoacridine linked platinum analogues to further characterise the binding efficiency of the drugs. The 9-aminoacridine analogues are intercalating agents with high affinity for DNA binding. These intercalator displacement assays have been used frequently to investigate other antitumour agents. This enabled us to calculate the relative DNA binding efficiency of cisplatin, the analogues of interest and other agents.This thesis also examined the extent of DNA unwinding by these analogues compared with cisplatin. The unwinding property was analysed by recording the change in electrophoretic mobility of open c

Bleomycin is an anti-tumour agent that is clinically used to treat several types of cancers. Bleomycin cleaves DNA at specific DNA sequences and recent genome-wide DNA sequencing specificity data indicated that the sequence 5'-RTGT*AY (where T* is the site of bleomycin cleavage, R is G/A and Y is T/C) is preferentially cleaved by bleomycin in human cells. Based on this DNA sequence, a plasmid clone was constructed to explore this bleomycin cleavage preference. By systematic variation of single nucleotides in the 5'-RTGT*AY sequence, this study was able to investigate the effect of nucleotide changes on bleomycin cleavage efficiency. It was observed that the preferred consensus DNA sequence for bleomycin cleavage in the plasmid clone was 5'-YYGT*AW (where W is A/T). The most highly cleaved sequence was 5'-TCGT*AT and the seven most highly cleaved sequences conformed to the consensus sequence 5'-YYGT*AW. A comparison with genome-wide results was also performed and while the core sequence was similar in both environments, the surrounding nucleotides were different.This study also examined the DNA sequence specificity of gamma radiation-induced DNA damage in purified DNA sequences. A comparison between the radiation-induced damage plasmid DNA and genome-wide human DNA data was studied. DNA was fluorescently end-labelled and gamma radiation-induced DNA cleavage was examined using capillary electrophoresis with laser-induced fluorescence detection. Previously, ionising radiation-induced DNA damage to end-labelled DNA has been reported to be non-sequence specific; however, this study provides an evidence that gamma radiation-induced DNA damage is sequence specific. The degree of cleavage was quantified at each nucleotide and it was observed that preferential cleavage occurred at cytosine nucleotides with lesser cleavage at guanine nucleotides, while being very low at thymine nucleotides. The differences in percentage cleavage at individual nucleotides ranged up to six-fold

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, was investigated in purified human mitochondrial DNA sequences using capillary electrophoresis with laser-induced fluorescence (CE-LIF). The drug was found to cleave preferentially at 5'-TGT*A-3' DNA sequences (where * is the cleavage site). In addition, a comparison was made between the bleomycin DNA sequence specificity obtained from genome-wide next-generation sequencing (NGS) with that obtained from purified DNA sequences. The overall bleomycin DNA sequence specificity was the same in the two environments, namely 5'-TGTA-3'. In this project, the sequence specificity of UV-induced DNA damage was also determined with a higher precision and accuracy than previously reported. UV light induces two major damage adducts: cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). Employing CE-LIF and taking advantages of the distinct properties of the CPDs and 6-4PPs, we studied the sequence specificity of UV-induced DNA damage in purified DNA sequences using two approaches: end-labelling and a polymerase stop/linear amplification assay. A mitochondrial DNA sequence that contained a random nucleotide composition and another synthetic sequence that contained specific sequences were employed as target sequences. In this project, we extended the UV sequence specificity to a hexanucleotide level from the previously reported dinucleotide and trinucleotide level. With the end-labelling technique (for 6-4PPs), the consensus sequence was found to be 5'-GCTC*AC (where C* is the breakage site); while with the linear amplification procedure, it was 5'-TCTT*AC. The influence of neighbouring nucleotides on the degree of UV-induced DNA damage was also examined. Finally, we attempted to study the genome-wide DNA sequence specificity of UV-induced damage in human cells using Illumina NGS technology. We investigated the usefulness of E. coli RecBCD (exonuclease V) as a prelimina

The antitumor activity of bleomycin is thought to arise from its ability to mediate DNA damage. Previous studies established GT and GC sequences as the major sites of bleomycin cleavage. Adjacent nucleotides are also known to subtly influence bleomycin preference for a particular site—however, this has yet to be characterised in detail. Furthermore, analogues of bleomycin often cleave at similar sites, but their relative preference can differ.This thesis aims to examine the extended sequence specificity of bleomycin—and compare it to related analogues. Understanding the subtle differences in the sequence specificity of the bleomycins could help elucidate their mechanism of action—and subsequently the rational design of more effective analogues.We utilised two different approaches—first, the RTGTAY plasmid construct was designed to contain 32 motifs with the sequence XXGTAX, where the X nucleotides were systematically varied to allow for subtle quantifications. Second, we utilised next-generation sequencing methods to detect bleomycin cleavage sites in the entire human genome—and in cellular environments. Previous studies often utilised short purified DNA sequences that did not permit the importance of complex cellular components to be evaluated. Using next-generation sequencing allowed us to examine bleomycin cleavage over a vast combination of DNA sequences—generating over 200 million cleavage sites per sample, enabling detailed characterisation of the bleomycin sequence specificity in human cellular and purified DNA environments.Utilising Illumina next-generation sequencing, we were able to rank DNA motifs based on their frequency of cleavage by bleomycin. We show that the strongest cleaved sites contained the core sequence TGTA. Interestingly, there were slight differences in the bleomycin specificity on purified genomic DNA, compared to cellular. Additionally, the results from the RTGTAY construct closely mirror that of the purified genomic DNA.We also utilised