Guiding strand passage: DNA-induced movement of the gyrase C-terminal domains defines an early step in the supercoiling cycle

Abstract

DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA in a strand passage mechanism. A double-stranded segment of DNA, the T-segment, is passed through the gap in a transiently cleaved G-segment by coordinated closing and opening of three protein interfaces in gyrase. T-segment capture is thought to be guided by the C-terminal domains of the GyrA subunit of gyrase that wrap DNA around their perimeter and cause a DNA-crossing with a positive handedness. We show here that the C-terminal domains are in a downward-facing orientation in the absence of DNA, but swing up and rotate away from the gyrase body when DNA binds. The upward movement of the C-terminal domains is an early event in the catalytic cycle of gyrase that is triggered by binding of a G-segment, and first contacts of the DNA with the C-terminal domains, and contributes to T-segment capture and subsequent strand passage.

Direct control of type IIA topoisomerase activity by a chromosomally encoded regulatory protein

“…6A,B; Kirchhausen et al 1985;Costenaro et al 2005;Lanz and Klostermeier 2011). Scattering data obtained for gyrase DCTD in the absence of YacG showed that the protein adopted an extended conformation with a significantly greater radius of gyration (R g ) than that seen for samples prepared with YacG (R g, gyrase = 90.3 Å 6 1.7 Å vs. R g, gyrase•YacG = 62.8 Å 6 2.7 Å ) (Fig.…”

The Acidic C-terminal Tail of the GyrA Subunit Moderates the DNA Supercoiling Activity of Bacillus subtilis Gyrase

“…The isolated CTD binds DNA via an elongated patch of positively charged amino acids around its perimeter, resulting in DNA bending and introduction of positive writhe (22,24). In gyrase, the CTDs associate with DNA adjacent to the G-segment that extends from the DNA-gate on the enzymatic core (26,27). Contact formation between these DNA regions and the CTDs is accompanied by a repositioning of the CTDs, aligning them with the DNA-gate (26).…”

“…In gyrase, the CTDs associate with DNA adjacent to the G-segment that extends from the DNA-gate on the enzymatic core (26,27). Contact formation between these DNA regions and the CTDs is accompanied by a repositioning of the CTDs, aligning them with the DNA-gate (26). The geometry imposed on the DNA by the CTDs directs the DNA exiting from the CTDs toward the N-gate in an orientation that allows the flanking region to serve as a T-segment for strand passage (15,26,28).…”

“…Contact formation between these DNA regions and the CTDs is accompanied by a repositioning of the CTDs, aligning them with the DNA-gate (26). The geometry imposed on the DNA by the CTDs directs the DNA exiting from the CTDs toward the N-gate in an orientation that allows the flanking region to serve as a T-segment for strand passage (15,26,28). Establishing this strand crossing is merely an effect of DNA binding and manifests in stabilization of a positive supercoil.…”

Potassium Ions Are Required for Nucleotide-induced Closure of Gyrase N-gate

“…causes the CTDs to move upward and sideways (9), and complete wrapping of DNA leads to narrowing of the N-gate formed by the GyrB subunits (8). The N-gate acts as an ATPdependent clamp (10): ATP binding to the ATPase domains of the GyrB subunits induces GyrB dimerization and N-gate closure (8,(11)(12)(13), leading to the trapping of the transport DNA (T-segment).…”