DAUNOMYCIN DISRUPTS NUCLEAR ASSEMBLY AND THE COORDINATE INITIATION OFDNA-REPLICATION IN XENOPUS EGG EXTRACTS

We have used Xenopus egg extracts to investigate the effects of the an titumor drug daunomycin on DNA replication in vitro. Xenopus sperm nuc lei replicated nearly synchronously in our egg extracts, thereby allow ing us to determine the effects of the drug on both replication initia tion and elongation. Titration experiments demonstrated that daunomyci n effectively inhibited replication in the extract, with 50% inhibitio n at a total drug concentration of 2.7 mu M. However, a high concentra tion of daunomycin (50 mu M) also inhibited nuclear envelope assembly, a prerequisite for the initiation of replication in this system. Ther efore, to bypass the effects of daunomycin on nuclear envelope assembl y, sperm nuclei were preassembled in extract prior to drug addition. I nitiation of replication in preassembled nuclei was also inhibited by daunomycin, with 50% inhibition at a drug concentration of 3.6 mu M. A t low drug concentrations, where replication did occur, the synchrony of initiations within individual nuclei was lost. This drug-induced di sruption of initiation events may provide important clues regarding th e mechanism(s) by which these events are coordinated in eukaryotic cel ls. Daunomycin also inhibited replication elongation in preassembled, preinitiated nuclei. However, the concentration of drug required for 5 0% inhibition of elongation was nearly fourfold higher than that requi red for inhibition of initiation. Taken together, these data demonstra te that Xenopus egg extract can be used to investigate the effects of DNA-binding antitumor drugs on a number of interrelated cellular proce sses, many of which are less tractable in whole cell systems. (C) 1997 Wiley-Liss, Inc.