title: Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage.
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.
authors.affiliation: Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas (CSIC), Barcelona 08028, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Martínez-García
authors.forename: Xavier
authors.initials: X
authors.lastname: Fernández
authors.forename: Ofelia
authors.initials: O
authors.lastname: Díaz-Ingelmo
authors.forename: Antonio
authors.initials: A
authors.lastname: Rodríguez-Campos
authors.forename: Chaysavanh
authors.initials: C
authors.lastname: Manichanh
authors.forename: Joaquim
authors.initials: J
authors.lastname: Roca
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D009690:Nucleic Acid Conformation
chemical_list: D004278:DNA, Superhelical; D004247:DNA; D004250:DNA Topoisomerases, Type II