The C-terminal domain of DNA gyrase A adopts a DNA-bending β-pinwheel fold

Abstract

DNA gyrase is unique among enzymes for its ability to actively introduce negative supercoils into DNA. This function is mediated in part by the C-terminal domain of its A subunit (GyrA CTD). Here, we report the crystal structure of this Ϸ35-kDa domain determined to 1.75-Å resolution. The GyrA CTD unexpectedly adopts an unusual fold, which we term a ␤-pinwheel, that is globally reminiscent of a ␤-propeller but is built of blades with a previously unobserved topology. A large, conserved basic patch on the outer edge of this domain suggests a likely site for binding and bending DNA; fluorescence resonance energy transfer-based assays show that the GyrA CTD is capable of bending DNA by >180°over a 40-bp region. Surprisingly, we find that the CTD of the topoisomerase IV A subunit, which shares limited sequence homology with the GyrA CTD, also bends DNA. Together, these data provide a physical explanation for the ability of DNA gyrase to constrain a positive superhelical DNA wrap, and also suggest that the particular substrate preferences of topoisomerase IV might be dictated in part by the function of this domain.T he topology of cellular DNA is managed by topoisomerases, enzymes that pass DNA strands through each other to relieve excess supercoiling and resolve DNA knots and catenanes (1, 2). Whereas all organisms contain at least one topoisomerase, the bacterium Escherichia coli possesses four, each with distinct roles: topoisomerase (topo) I, topo III, topo IV, and DNA gyrase. Topo I and topo III pass single DNA strands through one another to relax negative supercoils (1) or aid RecQ-family helicases in certain DNA repair processes, respectively (3). DNA gyrase and topo IV use ATP to power the transport of one intact DNA duplex through another, an activity that can alter DNA superhelicity as well as promote chromosome decatenation (4, 5).DNA gyrase and topo IV are members of the type IIA topoisomerase superfamily (6, 7). These enzymes are assembled as oligomeric complexes with distinct domains that coordinate ATP binding and hydrolysis with DNA binding, cleavage, and transport (7,8). The type IIA topo reaction cycle begins when one segment of DNA, termed the G segment, binds across the central region of the enzyme. ATP binding then triggers a series of motions that leads to the capture of a second DNA duplex (the T segment), cleavage and opening of the G segment, and passage of the T segment through the break. Once the T segment is transported, the G segment is resealed, the T segment is expelled from the protein, and ATP is hydrolyzed and released. This enzymatic cycle alters the linking number of the substrate DNA in discrete steps of Ϯ 2.Although all type IIA topos share this basic mechanism, there exist distinct type IIA subtypes that have differing substrate specificities and activities. The eukaryotic enzyme, topo II, relaxes positively and negatively supercoiled DNAs at the same rate (9) and can decatenate chromosomes (5). In contrast, most bacteria possess two somewhat more specialized type IIA topos; DNA ...

Structure of the Topoisomerase IV C-terminal Domain

“…Together, these results suggested that the DNA binding properties of these two prokaryotic enzymes are very different. It has been proposed that, with the help from GyrA-CTD, a continuous stretch of DNA can be wrapped by DNA gyrase to generate a (ϩ) supercoil-like intramolecular DNA cross-over to facilitate subsequent duplex passage reaction that produces net (Ϫ) supercoils in DNA (28,30,32). Consistent with its predicted catalytic function, deletion of GyrA-CTD converted DNA gyrase into a conventional type IIA Topo (26).…”

“…Recently, the crystal structure of GyrA-CTD from Borrelia burgdorferi has been determined (30). Although the structure was globally reminiscent of a six-bladed ␤-propeller as predicted (25), GyrA-CTD folds with a novel topology and was alternatively termed to adopt a ␤-pinwheel fold (30).…”

Ability of Viral Topoisomerase II To Discern the Handedness of Supercoiled DNA: Bimodal Recognition of DNA Geometry by Type II Enzymes

“…Previous studies with human and bacterial topoisomerases (19)(20)(21)(25)(26)(27)(28) suggest that the type II enzyme utilizes two distinct mechanisms to recognize the handedness of DNA supercoils. It has been proposed that the ability of some type II enzymes, such as human topoisomerase IIα and E. coli topoisomerase IV, to distinguish supercoil geometry during DNA relaxation is mediated by elements in the variable C-terminal domain (19,(25)(26)(27).…”

DNA topoisomerase II, genotoxicity, and cancer

“…However, several recent studies suggest that this portion of the protein plays an intriguing and important role in the recognition of DNA geometry [59][60][61][62][63][64][65]. As such, it may impart unique attributes, such as the ability to supercoil DNA [59][60][61] or act in front of replication forks [62,64], to specific type II enzymes. Unfortunately, no structural information is available for the C-terminal domain of any eukaryotic type II enzyme at the present time.…”

“…In contrast, topoisomerase IIβ, which is not believed to play a role in DNA replication, relaxes positive and negative superhelical twists at similar rates [62]. On the basis of amino acid sequence comparisons between the two human topoisomerase II isoforms, as well as studies on bacterial and viral type II enzymes [59][60][61]65], it has been proposed that the ability to discern the geometry of DNA supercoils during relaxation resides in the C-terminal domain of human topoisomerase IIα.…”