SmFRET Experiments Reveal Conformational Changes in DNA Gyrase that Drive Strand Passage Towards Negative Supercoiling

Abstract

DNA topoisomerases catalyze the inter-conversion of DNA topoisomers and impact replication, recombination, and transcription. DNA gyrase negatively supercoils DNA in an ATP-dependent process. Supercoiling occurs by a strand passage mechanism that requires the opening and closing of three transient gyrase interfaces, the N-gate, the DNA-gate, and the C-gate. The mechanism of coordination of these conformational changes is currently unknown.To dissect individual conformational changes and gain insight into their coordination, we monitored conformational changes of the gyrase DNA-gate, the N-gate, and the C-terminal domains (CTDs) of gyrase in single molecule FRET experiments with donor/acceptor labeled gyrase. In addition, we dissected the roles of DNA (G-segment) binding at the DNA-gate, wrapping by the CTDs, and capture of the transported DNA (T-segment) using linear DNAs of different lengths, and relaxed and negatively supercoiled plasmid DNA. We propose a detailed model for DNA supercoiling, in which the DNA bound at the DNA-gate is distorted and cleaved in a tightly coupled process. Flanking regions contact the CTDs, causing an upward movement. Upon complete wrapping of DNA, N-gate narrowing positions a T-segment in the upper cavity, and unlocks of the DNA-gate. ATP-binding then poises gyrase for strand passage: N-gate closure traps the T-segment, in a flip-flop mechanism triggers opening of the DNA-gate, and pushes the T-segment through the gap in the G-segment at the DNA-gate. DNA-gate closure renders strand passage irreversible, and the T-segment is released through the transiently opening C-gate. ATP hydrolysis, N-gate opening, and possibly CTD release complete the supercoiling cycle. Our results illustrate how a hierarchical, coordinated and tightly coupled sequence of conformational changes at the beginning of the supercoiling reaction strictly couples the nucleotide cycle to DNA strand passage in the catalytic cycle of gyrase.