Nucleic Acids Research, 1995, Vol. 23, No. 9 1524-1530
© 1995
CHEMISTRY |
Novel DNA photocleaving agents with high DNA sequence specificity related to the antibiotic bleomycin A2
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba Meguro-ku, Tokyo 153, Japan 1Faculty of Pharmaceutical Sciences, The University of Tokyo Hongo, Bunkyo-ku, Tokyo 113, Japan
*To whom correspondence should be addressed
Received January 23, 1995. Revised March 21, 1995. Accepted March 21, 1995.
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-nltrobenzoyl group co-valently 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 bithlazole 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 blthiazole derivatives bind and attack the minor groove of DNA. A photoactive unlthiazole 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-nrtrobenzoyl was linked to the intercalator acridlne 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 brthia-zole/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.