DNA knotting abolishes in vitro chromatin assembly.

Topological knots can be formed in vitro by incubating covalently closed double stranded DNA and purified topoisomerase II from the yeast Saccharomyces cerevisiae in an ATP-dependent reaction. Knotting production requires a starting enzyme/DNA mass ratio of 1. Analysis of knotted DNA was carried out by using both one- and two-dimensional agarose gel electrophoresis. The knots generated are efficiently untied, and give relaxed DNA rings, by catalytic amounts of topoisomerase II, but not by topoisomerase I. Time course analysis shows the knotting formation over relaxed and supercoiled DNA. When supercoiled DNA was used as a susbtrate, knots appear immediately whereas no transient relaxed rings were observed. The cell-free extract from Xenopus oocytes S-150 cannot assemble nucleosomes on knotted DNA templates as revealed by topological and micrococcal nuclease analysis. Nevertheless, the presence of knotted DNA templates does not inhibit the assembly over the relaxed plasmid. Finally, a pretreatment of knotted DNA with trace amounts of topoisomerase II before the addition of the S-150 yields a canonical minichromosome assembled in vitro. Taking into account these results, I suggest a mechanism of chromatin assembly regulation directed by topoisomerase II.