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Figure 1. Electrophoretic mobility of single DNA topoisomers containing different amount of UV photoproducts. Left: electrophoretic pattern on 1% agarose gel of plasmid pAT 153. The population of single topoisomers was obtained by exposure of naturally supercoiled DNA to Micrococcus luteus DNA topoisomerase I. Right: relative mobility of each visible band as a function of pyrimidine dimer content. RFI: fully supercoiled DNA; RFII: circular DNA containing at least one-single strand nick.

DNA topoisomerases catalyze the inter-conversion of DNA topoisomers and impact key cellular events such as replication, recombination, and transcription. Gyrase catalyzes the introduction of negative supercoils into DNA via a strand-passage mechanism. In the first step, a DNA-segment, the gate-DNA, binds to gyrase. The gate-DNA is cleaved, and a covalent DNA-gyrase complex is formed. A second DNA segment, the transfer-DNA, is passed through the gap, and the gate-DNA is re-ligated. Strand passage requires opening of a transient protein interface at the cleavage site, the so-called DNA-gate, by ∼2 nm. The intermediate cleavage complex presents an inherent danger of double strand DNA breaks and thus genome instability, and cleavage complexes have consistently been detected in very low amounts. In contrast, a recent study predicted frequent opening of the topoisomerase II DNA-gate. Here, we present a single molecule FRET study that monitors both the conformation of DNA bound to the DNA-gate of gyrase, and the conformation of the DNA-gate itself. DNA bound to gyrase adopts two different conformations, one slightly, one severely distorted from B-DNA geometry. Distortion requires cleavage, but neither ATP nor a transfer-DNA. The DNA-gate of gyrase is predominantly in the closed conformation, in agreement with