title: Novel DNA photocleaving agents with high DNA sequence specificity related to the antibiotic bleomycin A2.
abstract: We have designed and synthesized a series of novel DNA photocleaving agents which break DNA with high sequence specificity. These compounds contain the non-diffusible photoactive p-nitrobenzoyl group covalently linked via a dimethylene (or tetramethylene) spacer to thiazole analogues of the DNA binding portion of the antibiotic bleomycin A2. By using a variety of 5' or 3' 32P-end labeled restriction fragments from plasmid pBR322 as substrate, we have shown that photoactive bithiazole compounds bind DNA at the consensus sequence 5'-AAAT-3' and induce DNA cleavage 3' of the site. Analysis of cleavage sites on the complementary DNA strand and inhibition of DNA breakage by distamycin A indicates these bithiazole derivatives bind and attack the minor groove of DNA. A photoactive unithiazole compound was less specific inducing DNA breakage at the degenerate site 5'-(A/T)(AA/TT)TPu(A/T)-3'. DNA sequence recognition of these derivatives appears to be determined by the thiazole moiety rather than the p-nitrobenzoyl group: use of a tetramethylene group in place of a dimethylene spacer shifted the position of DNA breakage by one base pair. Moreover, much less specific DNA photocleavage was observed for a compound in which p-nitrobenzoyl was linked to the intercalator acridine via a sequence-neutral hexamethylene spacer. The 5'-AAAT-3' specificity of photoactive bithiazole derivatives contrasts with that of bleomycin A2 which cleaves DNA most frequently at 5'-GPy-3' sequences. These results suggest that the cleavage specificity exhibited by bleomycin is not simply determined by its bithiazole/sulphonium terminus, and the contributions from other features, e.g. its metal-chelating domain, must be considered. The novel thiazole-based DNA cleavage agents described here should prove useful as reagents for probing DNA structure and for elucidating the molecular basis of DNA recognition by bleomycin and other ligands.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Kuroda
authors.forename: H
authors.initials: H
authors.lastname: Satoh
authors.forename: M
authors.initials: M
authors.lastname: Shinomiya
authors.forename: T
authors.initials: T
authors.lastname: Watanabe
authors.forename: M
authors.initials: M
authors.lastname: Otsuka
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001761:Bleomycin / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000138:chemical synthesis* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D017421:Sequence Analysis
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D001761:Bleomycin