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Abstract
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 sequences surrounding the most intense IR-induced DNA cleavage sites were examined and a consensus sequence 5'-AGGC*C (where C* is the cleavage site) was found. The highest intensity gamma radiation-induced DNA cleavage sites were found at the dinucleotides, 5'-GG*, 5'-GC, 5'-C*C and 5'-G*G, and at the trinucleotides, 5'-GG*C, 5'-TC*A 5'-GG*G 5'-GC*C. These observations have implications for our understanding of ionising radiation-induced DNA damage.