Sequence-encoded DNA mechanics regulates binding and catalysis by DNA gyrase

Sequence-encoded DNA mechanics influences DNA:protein interactions, but whether it directly regulates the binding and catalytic activities of large DNA-remodellers remains unclear. DNA gyrase, an essential bacterial topoisomerase, negatively supercoils DNA by wrapping ~140 bp around two C-terminal domains (CTDs) before strand passage. Here we combine SELEX with high-throughput competitive binding and supercoiling assays to quantify how substrate sequence modulates gyrase binding and supercoiling activity. We interpret these sequence dependencies in terms of intrinsic cyclisability – a measure of mesoscale DNA deformability. DNA deformability within CTD-binding regions emerges as a key determinant of gyrase binding. However, supercoiling peaks at intermediate binding strength, revealing a trade-off between stable enzyme anchoring and conformational plasticity required for strand passage and supercoiling. Consistent with this trade-off, both high-affinity synthetic and native substrates display mechanical asymmetry, with flexible DNA primarily engaging one half of the enzyme footprint. Our measurements are consistent with a stable DNA:CTD interaction at one site compensating for weaker engagement at the second CTD. Finally, our results suggest that sequence-encoded DNA mechanics around promoters may influence gene expression by shaping local gyrase binding and supercoiling propensities. Overall, we establish DNA mechanics as an evolvable, sequence-dependent regulator of DNA remodeller activity.