Pulse-Labeling of Kinetoplast DNA: Localization of 2 Sites of Synthesis Within the Networks and Kinetics of Labeling of Closed Minicircles*

SYNOPSIS. Short pulse-labeling of log phase Crithidia fasciculata cells with ³H]thymidine allowed the autoradiographic visualization of 2 sites of replication of kinetoplast DNA situated at the periphery of the networks and separated by 180°. Longer pulse-labeling led to the previously reported total peripheral labeling pattern. Pulse-labeled networks possess an intermediate density in ethidium bromide-CsCl equilibrium gradients between the densities characteristic of closed networks and open or linear DNA. Removal of ethidium bromide by several methods and treatment of intermediate band networks with RNase and pronase had no effect on the equilibrium rebanding pattern. Closed minicircles of Leishmania tarentolae are not labeled by a short pulse of intact cells with ³H]thymidine. A chase of ～ 3–4 hr is required for the appearance of radioactivity in closed minicircles, a time delay which implies the existence of intermediate events between replication and eventual covalent closure of the minicircles.