Requirement of DNA topoisomerases for in vitro chromatin assembly by 3T6 mouse cell extracts.

Nuclear extracts of 3T6 mouse cells were able to assemble in vitro minichromosomes which displayed a 150-bp periodicity. Activities of both DNA topoisomerases I and II were detected in these extracts. When a supercoiled pUC DNA was added, it was first relaxed in less than 3 min, then slowly supercoiled again in 1-4 h. Both reactions occurred either in the absence or the presence of added Mg2+ and/or ATP, they were not blocked by DNA topoisomerase II inhibitors and they were inhibited by an antiserum against DNA topoisomerase I and by camptothecin. These findings led us to propose that, under our in vitro assay conditions, chromatin assembly is mainly carried out by a DNA topoisomerase I.