Sedimentation studies of the size and shape of DNA

Analytical zone sedimentation (Vinograd, Bruner, Kent & Weigle, 1963) is shown to be a sensitive and reliable method for detecting differences in conformation and for determining the molecular weight of homogeneous phage DNA's. Changes in sedimentation coefficient as a function of pH and ionic strength reveal transitions between native and denatured DNA and between different forms of denatured DNA. In alkaline solution, denatured DNA appears to exist in a random coil conformation, whereas at neutral pH it is contracted by intrastrand base-base interactions to a degree determined by the ionic strength. The denatured forms appear to be the single strands of the native DNA double helix. Calibrations of sedimentation coefficient against molecular weight are derived for single -stranded DNA in neutral and alkaline molar sodium chloride and for native DNA in molar sodium chloride. Degradation of homogeneous DNA is sensitively detected, and endonuclease I of Escherichia coli is shown to make double-strand breaks in native DNA, as distinct from pancreatic DNase which makes single-strand breaks.