title: The determination of the DNA sequence specificity of bleomycin-induced abasic sites.
abstract: The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, was determined with high precision in purified plasmid DNA using an improved technique. This improved technique involved the labelling of the 5'- and 3'-ends of DNA with different fluorescent tags, followed by simultaneous cleavage by bleomycin and capillary electrophoresis with laser-induced fluorescence. This permitted the determination of bleomycin cleavage specificity with high accuracy since end-label bias was greatly reduced. Bleomycin produces single- and double-strand breaks, abasic sites and other base damage in DNA. This high-precision method was utilised to elucidate, for the first time, the DNA sequence specificity of bleomycin-induced DNA damage at abasic sites. This was accomplished using endonuclease IV that cleaves DNA at abasic sites after bleomycin damage. It was found that bleomycin-induced abasic sites formed at 5'-GC and 5'-GT sites while bleomycin-induced phosphodiester strand breaks formed mainly at 5'-GT dinucleotides. Since bleomycin-induced abasic sites are produced in the absence of molecular oxygen, this difference in DNA sequence specificity could be important in hypoxic tumour cells.
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, 2052, Australia.
authors.forename: Jon K
authors.initials: JK
authors.lastname: Chen
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, 2052, Australia. v.murray@unsw.edu.au.
authors.forename: Vincent
authors.initials: V
authors.lastname: Murray
mesh_terms: D001483:Base Sequence; D001761:Bleomycin / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects* / Q000235:genetics*; D053837:DNA Cleavage; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015394:Molecular Structure; D013329:Structure-Activity Relationship
chemical_list: D001761:Bleomycin; D004247:DNA
keywords: Abasic DNA damage, bleomycin; Cleavage sites; DNA sequence specificity; End-label bias; Endonuclease IV