Hydroxyurea (HU) treatment of HSV-infected cells markedly inhibits the synthesis of virus DNA. Only 0.36% of the H-thymidine label was incorporated into virus DNA in the presence of HU as compared to untreated infected cells. Removal of HU resulted in a renewed synthesis of virus DNA as determined by the gradual increase in the incorporation of H-thymidine into HSV DNA. The labelled virus DNA molecules were isolated and chromatographed on benzoylated napthoylated DEAE (BND)-cellulose columns to separate the replicative intermediates that have single-stranded (ss) sequences from the mature double-stranded (ds) DNA genomes. Mature radioactive dsDNA molecules were found to appear at 22 min after removal of HU and gradually increased in amount thereafter. The virus DNA molecules synthesized during the initial 20 min after removal of HU, constitute the replicative intermediates of HSV DNA. It was calculated that the synthesis of HSV DNA proceeds at the rate of about 5 × 10 daltons per min.


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