Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes

Freshly prepared thymocytes continue to synthesize DNA under hypotonic conditions in the presence of 4.5% dextran T‐150, the four 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 approximately 30% of the rate in vivo. Synthesis in permeable cells reflects DNA replication shown by inhibitors such as 1‐β‐d‐arabinofuranosylcytosine 5′‐triphosphate (aCTP), nalidixie 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 [γ‐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.