Calf-Thymus Topoisomerase I Equilibrates Metastable Secondary Structure Subsequent to Relaxation of Superhelical Stress

Abstract

After relaxation of superhelical stress by various methods not involving topoisomerases, a long-lived metastable secondary structure with an anomalously low torsion elastic constant commonly prevails. The aim here is to ascertain whether such metastable secondary structure also results from the action of calf-thymus topoisomerase I (CT Topo I) on a native supercoiled DNA and, if so, whether the enzyme catalyzes its subsequent equilibration. The action of CT Topo I on supercoiled p30delta DNA was examined over a range of times from 10 min to 6 h. We verify that the enzyme operates in an almost completely processive manner, and at each time point determine the twist energy parameter, E(T), that governs the supercoiling free energy. E(T) is initially low, 533 +/- 60, and remains essentially constant up to at least 360 min, when no further CT Topo I is added. The activity of the rather dilute enzyme dies within approximately 60 min. During the 60 min after a second addition of fresh enzyme at either 60 or 120 min, E(T) rises up to a plateau at approximately 1100, which lies within the consensus equilibrium range, 1000 +/- 100. Over that same time period, the average peak spacing between the gel bands (corresponding to individual topoisomers) decreases somewhat with increasing time of exposure to active CT Topo I. After a third addition of fresh CT Topo I at 240 min, there is no further change in either E(T) or the average gel spacing. These and other observations indicate that active CT Topo I catalyzes the equilibration of a metastable secondary structure with abnormally low torsion and bending elastic constants that prevails after the initial release of superhelical stress. An observed temporal lag of this structural equilibration behind the relaxation of native superhelical DNAs suggests that it may require cleavage and religation events at multiple sites on the DNA. A novel analysis of the unwinding kinetics using literature data accounts for the almost complete processivity of the enzyme. The action of CT Topo I was also examined in the presence of 20 and 40 w/v% ethylene glycol (EG), which shift a secondary structure equilibrium toward an alternative state with altered torsion and bending elastic constants. The present results suggest that the usual metastable state coexists with the EG-induced state, and is equilibrated more rapidly than in the absence of EG.