Abstract
By transporting one DNA double helix (T-segment) through a double-strand break in another (G-segment), topoisomerase II reduces fractions of DNA catenanes, knots and supercoils to below equilibrium values. How DNA segments are selected to simplify the equilibrium DNA topology is enigmatic, and the biological relevance of this activity is unclear. Here we examined the transit of the T-segment across the three gates of topoisomerase II (entry N-gate, DNA-gate and exit C-gate). Our experimental results uncovered that DNA transport probability is determined not only during the capture of a T-segment at the N-gate. When a captured T-segment has crossed the DNA-gate, it can backtrack to the N-gate instead of exiting by the C-gate. When such backtracking is precluded by locking the N-gate or by removing the C-gate, topoisomerase II no longer simplifies equilibrium DNA topology. Therefore, we conclude that the C-gate enables a post-DNA passage proofreading mechanism, which challenges the release of passed T-segments to either complete or cancel DNA transport. This proofreading activity not only clarifies how type-IIA topoisomerases simplify the equilibrium topology of DNA in free solution, but it may explain also why these enzymes are able to solve the topological constraints of intracellular DNA without randomly entangling adjacent chromosomal regions.
Highlights
Type-IIA topoisomerases invert DNA crossovers by transporting one double helix (T-segment) through the transient double-strand break that they produce in another (G-segment) [1]
T2 was stored at a concentration of 2 mg/ml at À80C, and working stocks at 100 ng/ml were kept at À20C in 50 mM Tris-HCI, 1 mM ethylenediaminetetraacetic acid (EDTA), 500 mM KCI, 7 mM 2-mercaptoethanol, 100 mg/ml bovine serum albumin (BSA) and 50% (v/v) glycerol
Because the center of the non-equilibrium linking number topoisomers (Lk) distribution generated by T2 (LkS) did not always coincide with the equilibrium center (Lk0) produced by T1, LkS–Lk0 was defined as ÁLkS
Summary
Type-IIA topoisomerases (type-IIA) invert DNA crossovers by transporting one double helix (T-segment) through the transient double-strand break that they produce in another (G-segment) [1]. TypeIIA are homodimers of four functional domains: the ATP-ase domains or N-gate, the DNA cleavage-rejoining core or DNA-gate, the hinge domain or C-gate and the less-conserved C-terminal domains (CTDs) [1,2]. To catalyze DNA transport, a G-segment binds first to the cleavage-rejoining core to configure the DNA-gate [3]. Binding of ATP causes the ATPase domains to dimerize, and when this closure of the N-gate leads to the capture of a T-segment, a cascade of conformational changes ensues [3]. ATP hydrolysis starts during T-segment transport and concludes to allow N-gate reopening and enzyme turnover [7,8]
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