Analysis of Herpes Simplex Virus DNA Synthesized in Infected Nuclei by Chromatography on Benzoylated Naphthoylated DEAE Cellulose Columns

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Abstract
The nature of the DNA molecules synthesized in nuclei of herpes simplex virus (HSV)-infected [BSC-1 African Green monkey kidney] cells in vivo and in vitro was studied by chromatography on BND[benzoylated naphthoylated DEAE]-cellulose columns after shearing to DNA fragments of 10 to 20 .times. 106 daltons. The incorporation of labeled precursors occurs in the DNA fragments containing single-stranded regions, presumably by the replication forks. Prolongation of DNA synthesis leads to the accumulation of labeled DNA fragments that lack single-stranded sequences. Analysis of the isolated DNA fragments by density centrifugation in CsCl gradients revealed that most of the labeled DNA molecules are of virus specificity and the minority are cellular DNA fragments. Double-stranded virus DNA fragments and virus DNA fragments containing single-stranded sequences band in CsCl gradients at a density of 1.718 g/ml, the density of virion DNA. The replicating HSV DNA molecules may have the same density as the virion DNA and contain relatively little single-stranded DNA. The synthesis of HSV DNA molecules under in vitro conditions in isolated nuclei occurs by incorporation of the precursors into DNA fragments with single-stranded regions. The synthesis of cellular DNA in nuclei from hydroxyurea and cytosine arabinoside treated cells also occurs by elongation of nascent DNA chains.

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