Abstract
Gyrase is a type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme consists of two GyrA and two GyrB subunits. It is believed to introduce negative supercoils into DNA by converting a positive DNA node into a negative node through strand passage: First, it cleaves both DNA strands of a double-stranded DNA, termed the G-segment, and then it passes a second segment of the same DNA molecule, termed the T-segment, through the gap created. As a two-fold symmetric enzyme, gyrase contains two copies of all elements that are key for the supercoiling reaction: The GyrB subunits provide two active sites for ATP binding and hydrolysis. The GyrA subunits contain two C-terminal domains (CTDs) for DNA binding and wrapping to stabilize the positive DNA node, and two catalytic tyrosines for DNA cleavage. While the presence of two catalytic tyrosines has been ascribed to the necessity of cleaving both strands of the G-segment to enable strand passage, the role of the two ATP hydrolysis events and of the two CTDs has been less clear. This review summarizes recent results on the role of these duplicate elements for individual steps of the supercoiling reaction, and discusses the implications for the mechanism of DNA supercoiling.
Highlights
DNA supercoiling affects DNA replication and recombination, and gene expression [2,3]
This enzyme can introduce negative supercoils into DNA in steps of two with just a single C-terminal domains (CTDs) [40]. These results suggest that supercoiling by a minimal gyrase with one functional ATPase domain, only one CTD, and a single catalytic tyrosine is possible
DNA supercoiling by gyrase is supported by hydrolysis of a single ATP
Summary
DNA supercoiling affects DNA replication and recombination (reviewed in [1]), and gene expression [2,3]. WWrraappppiinngg ooff tthhee DDNNAA aarroouunndd ggyyrraassee iinn aa ppoossiittiivvee nnooddee bbyy mmeeaannss ooff tthhee CCTTDDss ((sseeee FFiigguurree 22)) [[3300,,3311]] ppoossiittiioonnss tthhee GG-aanndd TT--sseeggmmeennttss ffoorr ssttrraanndd ppaassssaaggee [[3322]]. The supercoiling efficiency of gyrase with a single functional ATPase domain is reduced compared with wildtype gyrase capable of binding and hydrolyzing two ATP molecules [41] This difference in activities suggests that gyrase catalyzes sequential hydrolysis of the two ATP molecules bound, as previously shown for topo II, and implies that an ATP/ADP·Pi-bound state is populated during the catalytic cycle and is functionally relevant. Gyrase with a single CTD shows DNA-induced N-gate narrowing and DNA-stimulated ATP hydrolysis [42], demonstrating that inter-domain communication is intact Most importantly, this enzyme introduces negative supercoils in steps of two. Gyrase with a single CTD behaves very similar to gyrase with two CTDs, whereas gyrase lacking both CTDs is an entirely different enzyme that catalyzes different reactions
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