Promoter-specific synthetic photoendonuclease: rose bengal-labeled T7 RNA polymerase.
We have constructed a synthetic photoendonuclease composed of T7 RNA polymerase linked to rose bengal. The promoter-specific polymerase confers site-specific binding, and the photosensitizer rose bengal allows light-induced DNA cleavage. Using a gentle labeling procedure, we find that the polymerase can be labeled with 1-30 rose bengals. Polymerase labeled to about 8 rose bengals per molecule retains the same efficiency and specificity of binding to promoter-containing DNA as unlabeled polymerase. At this level of rose bengal substitution, the synthetic endonuclease, in the presence of visible light, specifically cleaves linear or supercoiled DNA containing a T7 promoter. It induces frank single-strand breaks, rather than labile sites convertible to breaks upon additional treatments. Neither the free rose bengal moiety not bonded to polymerase nor the free (not bound to DNA) rose bengal-substituted polymerase cleaves DNA. Although rose bengal is an efficient generator of singlet oxygen, depletion of oxygen from reaction mixtures increases the cleavage rate. This indicates that singlet oxygen cleavage is not a major mechanism of DNA nicking by the synthetic endonuclease. At higher levels of rose bengal substitution, the labeled polymerase shows decreased binding efficiency and increased nonspecific binding to DNA without a T7 promoter; the specificity of DNA cleavage also decreases. These results indicate that the site specificity of rose bengal photocleavage by the synthetic endonuclease results from specific binding of the polymerase, and thus rose bengal photonicking reflects polymerase binding.
Sutherland
B M
BM
Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
Randesi
M
M
Wang
K
K
Conlon
K
K
Epling
G A
GA
D001665:Binding Sites; D004278:DNA, Superhelical / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008027:Light; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D010777:Photochemistry; D017319:Photosensitizing Agents / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012395:Rose Bengal / Q000494:pharmacology; D026082:Singlet Oxygen; D013604:T-Phages / Q000235:genetics*; D014764:Viral Proteins
D004278:DNA, Superhelical; D004279:DNA, Viral; D017319:Photosensitizing Agents; D014764:Viral Proteins; D026082:Singlet Oxygen; D012395:Rose Bengal; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D010100:Oxygen