Photochemical cross-linking of DNA-RNA helices by psoralen derivatives

Abstract

Bacteriophage fd DNA-RNA hybrid has been prepared in vitro by transcribing single-stranded fd DNA with Escherichia coli RNA polymerase. Most of the newly synthesized RNA was found to be associated with DNA on the basis of buoyant density studies in Cs 2SO 4 density gradients. First, the buoyant density of the complex increases with the extent of transcription. Secondly, the native complex is resistant to pancreatic RNAase digestion in 0.2 m-NaCl (37 °C, 30 min), whereas RNA of the heat-denatured complex can be hydrolyzed by the enzyme under the same conditions. The ability of psoralens to covalently cross-link DNA-RNA double helices has been tested by irradiating either 3H-labeled fd DNA- 32P-labeled RNA hybrid or unlabeled fd DNA- 32P-labeled RNA hybrid with long wavelength ultraviolet light in the presence of one of the three psoralen derivatives: 4′-aminomethyl-4,5′,8-trimethylpsoralen hydrochloride, 4′-hydroxymethyl-4,5′,8-trimethylpsoralen, or 4,5′,8-trimethylpsoralen. While the RNA and DNA strands of the native complex separate irreversibly after denaturation with heat or heat plus urea, the psoralen + u.v.-treated hybrids undergo a reversible helix-coil transition as shown by equilibrium Cs 2SO 4 density gradient ultracentrifugation, gel electrophoresis, and RNAase digestion experiments. These results indicate that covalent bridges form between the RNA and DNA strands after the photochemical reaction of the hybrid with AMT, HMT or TMP. The photochemically cross-linked DNA-RNA complexes have been denatured completely using the 37 °C (formaldehyde + formamide) technique (Shen & Hearst, 1977 b) and spread for electron microscopy to examine the interstrand cross-links directly. The average number of cross-links was calculated for these looped and tailed hybrid molecules and compared to that of TMP cross-linked and denatured double-stranded fd DNA restriction fragments. From the denaturation microscopy studies, it has been concluded that: (1) under the conditions of low cross-linking (9 μ m-base-pairs, 0.045 μ m-psoralen molecules, 10 m m-Tris · HCl, 1 m m-EDTA, 10 m m-NaCl, pH 8.0, 15 °C), the AMT, HMT and TMP hybrids have 2.0, 2.2 and 2.2 cross-links per molecule, respectively; (2) as the salt is increased to 200 m m-NaCl, the average number of the cross-links decreases to 0.7, 0.9 and 1.5; (3) TMP cross-links DNA helices more efficiently than DNA-RNA helices by factors of approximately 4 and 6 in 10 m m and 200 m m-NaCl, respectively; (4) the photochemical cross-linking reaction can be driven by higher concentrations of psoralens. With an initial concentration of 0.45 μ m-psoralen, the distance between adjacent cross-links decreases appreciably and many apparent double-stranded segments are observed on the denatured molecules. After the psoralen concentration is increased to 9 μ m, loops or tails are seen on only a few of the molecules, indicating that the DNA-RNA hybrid becomes densely cross-linked. The photochemical cross-linking reaction of DNA-RNA hybrid with psoralens should become a powerful tool in studying chromosome structure and genetic activities involving “transient” DNA-RNA hybrids.