The DNA sequence specificity of ultraviolet light and the anti-tumour drug bleomycin

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 preliminary step in genome-wide DNA sequencing. The enzyme was found to be inhibited by UV-induced DNA adducts and hence, it could be employed to reduce the large number of DNA sequences and enrich adduct-containing DNA sequences for NGS sequencing