Single-strand breaks in duplex DNA of coliphage T7 as demonstrated by electron microscopy

Abstract

Previous work based on ultracentrifugal studies had led to the hypothesis that there are either gaps or exceptional bonds, not of the phosphodiester type, in the single polynucleotide strands of the DNA of coliphage T7. To investigate this further, the length distribution of the single strands derived from native T7 DNA by alkaline denaturation has been obtained. An improved technique for electron microscopic visualization of single strands of DNA in a cytochrome c monolayer was used for this measurement. In contrast with the narrow length distribution for native, double-stranded DNA, the length distribution of the single strands was found to be very broad (about eightfold greater than for the native material). Single-stranded T7 DNA was then fractionated according to sedimentation coefficient by sucrose gradient centrifugation, and these various fractions were examined with the electron microscope. It was found that the more rapidly and more slowly moving fractions consist of longer and shorter filaments, respectively, from which it has been concluded that sedimentation heterogeneity of single-stranded DNA is a reflection of molecular weight heterogeneity. Hence, the electron microscopic studies confirm the original hypothesis that T7 DNA contains single-strand breaks or exceptional bonds.