Abstract
Type IIA topoisomerases (Top2s) manipulate the handedness of DNA crossovers by introducing a transient and protein-linked double-strand break in one DNA duplex, termed the DNA-gate, whose opening allows another DNA segment to be transported through to change the DNA topology. Despite the central importance of this gate-opening event to Top2 function, the DNA-gate in all reported structures of Top2-DNA complexes is in the closed state. Here we present the crystal structure of a human Top2 DNA-gate in an open conformation, which not only reveals structural characteristics of its DNA-conducting path, but also uncovers unexpected yet functionally significant conformational changes associated with gate-opening. This structure further implicates Top2’s preference for a left-handed DNA braid and allows the construction of a model representing the initial entry of another DNA duplex into the DNA-gate. Steered molecular dynamics calculations suggests the Top2-catalyzed DNA passage may be achieved by a rocker-switch-type movement of the DNA-gate.
Highlights
Type IIA topoisomerases (Top2s) manipulate the handedness of DNA crossovers by introducing a transient and protein-linked double-strand break in one DNA duplex, termed the DNA-gate, whose opening allows another DNA segment to be transported through to change the DNA topology
Type IIA topoisomerases (Top2s) are essential DNAmanipulating enzymes ubiquitous in eukaryotes and bacteria[1,2,3]. These enzymes exploit protein conformational changes driven by ATP binding and hydrolysis to direct the crossing of two DNA duplexes, leading to topological inversion of DNA crossovers[2,4]
The enzyme is reset for its catalytic cycle by subsequent steps[3,4,6], which include the release of the T-segment through the C-gate formed by the helical domain appended to the coiled-coil linker, religation of the cleaved G-segment by reversal of the transesterification reaction, and re-opening of the N-gate dimer interface upon ATP hydrolysis
Summary
Type IIA topoisomerases (Top2s) manipulate the handedness of DNA crossovers by introducing a transient and protein-linked double-strand break in one DNA duplex, termed the DNA-gate, whose opening allows another DNA segment to be transported through to change the DNA topology. Type IIA topoisomerases (Top2s) are essential DNAmanipulating enzymes ubiquitous in eukaryotes and bacteria[1,2,3] These enzymes exploit protein conformational changes driven by ATP binding and hydrolysis to direct the crossing of two DNA duplexes, leading to topological inversion of DNA crossovers[2,4]. We report the high-resolution view of the opening of the Top[2] DNA-gate, which directly mediates the resolution of topological strand crossings This structure reveals the formation and structural features of the T-segmentconducting path, and uncovers unexpected, functionally relevant, conformational changes that accompany the closedto-open transition. Using this structure as a starting point, we simulated the passage of the T-segment through the DNA-gate by steered molecular dynamics (SMD) simulations, to gain further insight into this central yet elusive step in Top[2] catalysis
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