The DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction

Abstract

Gyrase is the only type II topoisomerase that introduces negative supercoils into DNA. Supercoiling is catalyzed via a strand-passage mechanism, in which the gate DNA (gDNA) is transiently cleaved, and a second DNA segment, the transfer DNA (tDNA), is passed through the gap before the gDNA is religated. ATPase ͉ negative supercoiling ͉ topoisomerase ͉ single molecule FRET D NA topoisomerases modulate DNA structure by interconverting different DNA topoisomers (1). Gyrases are bacterial type II topoisomerases that use the chemical energy of ATP hydrolysis to introduce negative supercoils into DNA (2). The active form of gyrase is a heterotetramer formed by two GyrA and two GyrB subunits. Supercoiling is catalyzed via a strand-passage mechanism and involves the coordinated opening and closing of 3 different protein interfaces or gates, termed N-gate, DNA-gate, and C-gate (3-5) (Fig. 1A). As a first step, one segment of double-stranded DNA, the gate DNA (gDNA), binds to the DNA-gate formed by the GyrA subunits (6) that harbor the catalytic tyrosines (7). The transesterification reactions lead to the formation of a covalent protein-DNA intermediate (8). The N-gate is formed by the GyrB subunits, which dimerize in the presence of ATP and trap the transfer DNA (tDNA) segment (9-12). The tDNA can then pass through the gap created in the gDNA. After religation of the gDNA, the tDNA leaves the enzyme through the C-gate formed by the GyrA subunits (13,14). A concerted opening and closing of these gates is a prerequisite for tight coupling of DNA cleavage and strand passage during the supercoiling reaction. The so-called double lock rule has been proposed as a simple principle to ensure unidirectional strand passage in type II topoisomerases (15, 16) (19) and that have provided strong evidence that a tDNA is required for opening of the DNA-gate (20)(21)(22). Here, we present smFRET experiments that resolve this issue by directly monitoring the conformational state of the DNA-gate in Bacillus subtilis gyrase bound to dsDNA and during the relaxation and supercoiling reactions. These FRET experiments demonstrate that the DNA-gate is closed in the presence of linear dsDNA and during DNA relaxation or ATPdependent DNA supercoiling. Our results are in agreement with a severe distortion of gDNA coupled to DNA cleavage. DNA binding, distortion, and cleavage are mechanistically distinct steps that precede opening of the DNA-gate. Gate opening is a rare event during relaxation and supercoiling that only occurs briefly to allow for strand passage. Results A Small dsDNA Substrate Functions as the gDNA for B. subtilis Gyrase.Gyrases exhibit limited sequence specificity for DNA cleavage. To facilitate studies on the DNA gate conformation by FRET, we designed a 60-bp substrate for gyrase that contains a preferred cleavage site in the center (23), flanked by a donor (A488) and acceptor fluorophore (A546 or A555) (Fig. 1B). Cleavage at the preferred site would produce two fragments with 24/28 and 36/32 bases. When the anisotropy of A546 was...

DNA-induced narrowing of the gyrase N-gate coordinates T-segment capture and strand passage

“…S3) that contain a preferred cleavage site for B. subtilis gyrase (31) in the center. Binding of these DNAs to gyrase and cleavage at the preferred site were confirmed (13) (Fig. 4 A and B).…”

“…We have previously established that a linear DNA of 60 bp (Fig. S3) is a suitable model for a gate DNA (13). Interestingly, binding of this DNA had no effect on N-gate conformation (Fig.…”

“…The active form of DNA gyrase is a heterotetramer of two GyrA and two GyrB subunits. B. subtilis GyrA is a stable dimer, even at picomolar concentrations (13), whereas isolated GyrB is monomeric (21). To prevent formation of incomplete complexes (22,23) or complex dissociation during smFRET experiments that address conformational changes of the N-gate formed by the GyrB subunits, we fused the coding regions for GyrB and GyrA, such that the two subunits are connected via a linker of the sequence G-A-P (Fig.…”

“…The T-segment can pass the gap in the G-segment created by double-strand cleavage. Importantly, the DNA-gate is predominantly closed during negative supercoiling (13), indicating that opening of the gate occurs only transiently to allow for strand passage. An active contribution of the T-segment to the opening of the DNA-gate has been proposed (14)(15)(16).…”

“…Cleavage of this gate DNA (G-segment) generates a gap through which a second DNA segment (T-segment) is transported. We have previously shown that DNA bound to the DNA-gate is distorted in a process coupled to cleavage (13). The N-gate formed by the GyrB subunits acts as an ATP-dependent clamp that closes due to nucleotide-induced dimerization, leading to the trapping of a T-segment (4,14) in the upper cavity delimited by the N and the DNA-gate.…”