Studies on DNA topoisomerase activity during in vitro chromatin assembly

The in vitro assembly of chromatin, promoted by the Xenopus cell-free extract (S-150), can be inhibited by oxolinic acid and to a lesser extent by nalidixic acid. Both of these antibiotics have been shown to block the activity of the specialized type II Topoisomerase, bacterial DNA Gyrase. Oxolinic acid induces a DNA cleavage by Micrococcal Nuclease at specific sequences in the multiple cloning vector pGEM-4. Nalidixic acid does not inhibit DNA supercoiling, but does diminish the extent of chromatin formation achieved by the S-150 on circular DNA templates. The Topoisomerase I inhibitor, berenil, does not inhibit extensive chromatin assembly, although it does diminish the level of supercoiling. Taken together, these results suggest that both topoisomerases play a role in the assembly process. Topoisomerase I may catalyze both the introduction of unconstrained supercoils into relaxed DNA and the formation of monosomes, while Topoisomerase II may promote extended chromatin assembly.