Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes

Abstract

Freshly prepared thymocytes continue to synthesize DNA under hypotonic conditions in the presence of 4.5% dextran T-150, the 4 deoxyribonucleoside triphosphates and ATP. Permeable cells could seal the membrane in a serum-enriched medium within a few hours. 2''-Deoxycytidine 5''-triphosphate is effectively substituted by 5-mercuri-2''-deoxycytidine 5''-triphosphate as a substrate. The newly synthesized mercurated DNA can be separated from cellular DNA and RNA on a thiol-agarose affinity matrix. The rate of incorporation of [3H]thymidine triphosphate into permeable cells is the same as that of the incorporation of [3H]thymidine into intact cells, corresponding to .apprx. 30% of the rate in vivo. Synthesis in permeable cells reflects DNA replication shown by inhibitors such as 1-.beta.-D-arabinofuranosylcytosine 5''-triphosphate (aCTP), nalidixic acid and novobiocin, and by density shift experiments. More than 80% of the newly synthesized low-molecular-mass, DNA, 8-60 nucleotides in length, consists of RNA-linked DNA. This conclusion is based on phosphorylation with [.gamma.-32]ATP and polynucleotide kinase and rephosphorylation after alkaline hydrolysis. The 5'' end of RNA consists of adenylate, guanylate, cytidylate and uridylate residues in a ratio of 4:3:1.5:1.5.