Annealing of alkali-resistant HSV DNA strands and isolation of S and L components.

DNA isolated from highly purified virions of herpes simplex virus type-1 (HF strain) was denatured by centrifugation in alkaline sucrose gradients. DNA molecules corresponding to intact single-stranded virion DNA (50 x 10(6) daltons) were isolated and adjusted to neutral pH. The DNA was annealed under conditions permitting reassociation of intact single-stranded molecules and studied by electron microscopy. Three classes of DNA molecules showing double-stranded sequences were observed: (a) fully double-stranded DNA molecules the size of the intact HSV DNA genome, namely 52 micron; (b) DNA hybrids with a region of partial double-strandedness ranging from 5 to 12 micron, plus long single strands; and (c) DNA hybrids with a double-stranded region of 32--40 micron, plus short single strands. (These results suggest that the alkali-resistant single-stranded HSV DNA molecules are composed of several subclasses that permit annealing of either the total genome or the S or L components.) The 5 micron double-stranded region probably constitutes the S component of HSV DNA and the sequences longer than 5 micron and shorter than 12 micron represent annealing of the repeat sequences on either or both sides of the S component. The double-stranded sequences with a length of 32--40 micron may represent the L component. Treatment of the annealed, partially double-stranded hybrid DNA molecules with S1 endonuclease to remove the single-stranded termini and centrifugation in neutral sucrose gradients yielded two distinct peaks. Centrifugation of fractions from the two peaks in caesium chloride density gradients showed that the small DNA component (possibly the S and the repeat sequences) had a higher buoyant density and the longer (possibly the L) DNA component had a lower density than the HSV DNA marker. Annealing of alkali-resistant viral DNA strands therefore provides a means of isolating the L, S and repeat sequence regions of HSV DNA.