Catenation of DNA rings by topoisomerases. Mechanism of control by spermidine.

Abstract

Polyvalent cations such as spermidine are required for catenation (interlocking) of DNA rings by topoiso-merases. We show that this is due to compaction of the DNA substrate into aggregates where the high local DNA concentration favors the catenated state. The same critical concentration of polycations induced ag-gregation and catenation. They remained coincident when the critical threshold was increased by competing mono-or divalent cations and when dissimilar topoi-somerases (DNA gyrase and u protein) and polycations (spermidine and Co3+ (NH&) were used. Under disag-gregating conditions, topoisomerases unlinked cate-nanes. Topoisomerases allow DNA segments to pass through each other causing continual catenation and decatenation; the equilibrium position is dictated by the counterion composition. The aggregates were huge, sedimenting at 30,000 S, but the component DNA rings bound gyrase as well as dispersed rings. Aggregates formed and dispersed rapidly and freely exchanged rings with neighboring aggregates. The aggregates are dynamic structures accessible to enzymes and are a model for compacted DNA in vivo. We used an extension of the counterion condensation theory of Manning to calculate the charge of DNA in the presence of three cations of different valence, as in topoisomerase reactions. DNA rings cooperatively ag-gregated when about 90% of DNA charge was neutralized , in agreement with previous studies of the effect of two cations on intramolecular DNA compaction. We suggesl several tests of whether a polyamine effect is due to compaction, including a phase diagram showing the predicted ionic conditions for compaction. Application of these criteria to published data revealed that DNA compaction by polyamines is important in enzyme systems where distant DNA segments interact, such as site-specific recombination of bacteriophage X. Polyamines such as spermidine are ubiquitous: they are present in millimolar concentrations in bacteria, viruses, and actively proliferating animal cells (1-3). They dramatically affect the structure of nucleic acids (4,5) and many enzymatic reactions (l), and interruption of polyamine synthesis by mutations (6) or inhibitors (7) results in reduced or no cell growth. Rarely, however, is the role of the polyamine understood. In this report we defiie the precise role of polyamines in reactions of DNA topoisomerases. Topoisomerases cataiyze diverse reaction including the catenation (interlocking) and decatenation of duplex DNA rings, relaxation (removal) of supercoils, and in the unique case of DNA gyrase, introduction of supercoils in the presence of ATP (reviewed in Refs. 8 to 10). In all these reactions, topoisomerases introduce a transient break in a DNA segment, …