Abstract
Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA through a transient break formed in another duplex. The bacterial DNA gyrase, a target for broad-spectrum antibiotics, is the sole Topo2A enzyme able to introduce negative supercoils. We reveal here for the first time the architecture of the full-length Thermus thermophilus DNA gyrase alone and in a cleavage complex with a 155 bp DNA duplex in the presence of the antibiotic ciprofloxacin, using cryo-electron microscopy. The structural organization of the subunits of the full-length DNA gyrase points to a central role of the ATPase domain acting like a ‘crossover trap’ that may help to sequester the DNA positive crossover before strand passage. Our structural data unveil how DNA is asymmetrically wrapped around the gyrase-specific C-terminal β-pinwheel domains and guided to introduce negative supercoils through cooperativity between the ATPase and β-pinwheel domains. The overall conformation of the drug-induced DNA binding–cleavage complex also suggests that ciprofloxacin traps a DNA pre-transport conformation.
Highlights
The essential function of DNA topoisomerases regulates the underwinding or overwinding of DNA that occurs during replication, chromosome segregation or transcription of the intertwined DNA double helix [1,2]
Our cryo-electron microscopy (cryo-EM) data reveals for the first time the full architecture of DNA gyrase in absence and in presence of a 155 bp long DNA template, a structural state closer to the physiological context than previously observed in truncated enzyme with 20–34 bp DNA sequences
The projected path of the DNA suggests that the b-pinwheels position the T-segment toward the N-gate entry to form an angle of 60 with the G-segment, compatible with the proposed geometry of positive DNA crossover in the DNA-gate leading to negative supercoiling after strand passage (Figure 4A and 4C) [20,23]
Summary
The essential function of DNA topoisomerases regulates the underwinding or overwinding of DNA that occurs during replication, chromosome segregation or transcription of the intertwined DNA double helix [1,2] To help overcome these topological problems, type 2A DNA topoisomerases (Topo2A) bind and cut the phosphate backbone of double-stranded DNA (dsDNA). This transient break allows the DNA to be relaxed or untangled with resealing at the end of the process [2]. The eukaryotic Topo2A are homodimers in which the composing subunits correspond to a fused version of the B/E and A/C homologs into a single polypeptide [5] These modular enzymes possess a core set of conserved ATPase (N-gate), DNA binding–cleavage domains (DNA- and C-gate) and an idiosyncratic C-terminal domain (CTD; Figure 1A). Transport of the T-segment through the G-segment double strand break is coupled to ATP hydrolysis through coordinated opening and closing of the dimer interface in the so-called ‘three-gate mechanism’ that leads to the exit of the T-segment through the C-gate after G-segment religation [8,9]
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