Strand separation of DNA induced by ultraviolet irradiation in vitro

Abstract

1. Ultraviolet irradiation of double stranded DNA in vitro caused strand separation, as judged by the susceptibility of the DNA to degradation by an endonuclease from Neurospora crassa that is specific for single strands, by its behavior on elution from hydroxylapatite and by its sedimentation properties. The extent of degradation by the endonuclease was almost linearly proportional to the ultraviolet dose up to 2.8 · 10 5 J/m 2, when 45% of the DNA was degraded. Increased DNA concentration and high concentrations of Na + and Mg 2+ were inhibitory to degradation. The pH of the DNA solution had no significant effect. 2. The ultraviolet-irradiated DNA was resistant to single strand-specific exonuclease I from Escherichia coli even after removal of 5′-phosphate termini or limited endonucleolytic cleavage. Ultraviolet-induced denaturation was not reversible even under optimum conditions. 3. The elution profile of irradiated DNA from hydroxylapatite was gradually shifted, depending on the ultraviolet dose, from the position of double stranded DNA toward that of single stranded DNA, although there was no significant overlap with the profile of single stranded DNA. The elution profile also indicated that the denaturation was not an all-or-none phenomenon and that all the DNA molecules were similarly affected. Unlike X-irradiation, ultraviolet light did not generate single stranded fragments. 4. The sedimentation coefficient of T7 phage DNA increased from 31 to 67 S after ultraviolet irradiation (1.4 · 10 5 J/m 2 incident dose), presumably due to localized collapse of the double stranded structure and formation of interstrand cross-links. 50% of the total irradiated DNA was rapidly renaturable after denaturation by alkali. 5. Localized strand separation in DNA after ultraviolet irradiation was related to pyrimidine dimer formation; proflavine reduced both. The denatured regions contained almost all of the pyrimidine dimers. When the irradiated DNA was treated with N. crassa endonuclease, thymine-containing dimers were released as acid-soluble products more readily than was thymine. 6. DNA inside T7 phage was also denatured after ultraviolet irradiation of the whole phage. Such DNA was not denatured by alkali, and was covalently cross-linked with the phage coat.