Iodination of DNA. Studies of the reaction and iodination of papovavirus DNA

Abstract

Iodination of DNA by the reaction originally described by S. L. Commerford ((1971), Biochemistry 10, 1993) is extremely sensitive to the secondary structure of the DNA. Cytidines in denatured simian virus 40 (SV40) DNA react at a slightly slower rate than free cytidine monophosphate; hydrogen-bonded cytidines in SV40 form I DNA are iodinated considerably more slowly; elimination of the negative supercoils in form I DNA by conversion to form II or form III reduces reactivity even further. The residual reactivity of form II or form III duplex DNA is not due to preferential iodination of unpaired cytidines near phosphodiester bond breaks; rather iodination occurs throughout the molecule. Cytidine monophosphate has been used as a model for DNA, to enable spectral measurements of its reaction with iodine and T1C13. At temperatures above 42 degrees C and at pH 5.0, formation of 5-iodocytidine is limited by the rate of formation of an intermediate, probably 5-iodo-6-hydroxydihydrocytidine. At lower temperatures, the conversion of intermediate to product is rate limiting, but can be accelerated by lowering the pH. By appropriate adjustment of pH, or temperature, the formation of intermediate or its conversion to product can be accelerated. Iodination destabilizes the DNA duplex. Iodocytosines in SV40 DNA are preferentially removed by S1 nuclease. Heavily iodinated DNA does not reassociate normally, but DNA with only 5-10% of its cytosines iodinated appears to reassociate with normal kinetics, if duplex formation is measured by hydroxylapatite chromatography. Conditions are described to permit preparation of DNA, which reassociates normally, having a specific activity of 10(8) cpm/mug.