#catenane

Science, Volume 389, Issue 6761, Page 708-710, August 2025. Summary Without knowing the specific content of the Science article from August 2025, I can only offer a general summary. Assuming it’s a typical research article in Science, it likely presents novel findings in a scientific field. The article probably outlines a specific research question, the methodology used to investigate it, and the…

Science, Volume 389, Issue 6759, Page 526-531, July 2025. Summary Without knowing the specific article within Science, Volume 389, Issue 6759, pages 526-531 from July 2025, a summary is impossible. I need the article title or subject matter to provide an accurate 100-word summary. Please provide more information about the article content. Read more… Credits: Source This post is part of…

read at

http://www.rsc.org/chemistryworld/2015/01/rotaxane-catenane-transformation-light

The mechanism of negative DNA supercoiling

A cascade of DNA-induced conformational changes prepares gyrase for strand passage

A review came out this morning from the frontiers of a fascinating field in modern molecular biology — one that asks how DNA shapes up in the living organism. Yes, it forms a double helix (usually) but what happens then?

One way in which DNA is regulated at a biophysical level is through supercoiling — positive or negative — meaning twisting up to a greater or lesser extent. The name “supercoiling” highlights the fact that this is “on top of” the molecule's natural helical coil.

As with the stretching of other biomolecules, this action can expose binding sites (for transcription factors) and is crucial in regulating DNA replication, where negative supercoiling is required to allow processes such as replication, transcription and recombination to be carried out on the genome.

Coiling can only take place on a molecule that is either a closed loop (such as bacterial plasmids or DNA minicircles) or linear molecules that are fixed at certain points, such as by interacting proteins — making it for all intents and purposes the topological equivalent to a closed loop, and twist (or writhe) being converted to tension within the molecule rather than spinning motion.

The catenane in the centre of the simple diagram here clearly requires a separation (by a topoisomerase) to get back to the circular, relaxed form. Likewise, the positive and negative values of supercoiling are determined by whether the gyrases apply tension with or counter to the turn of the α-helix. Negative supercoiling (-sc) favours strand separation, while +sc stabilises double-stranded DNA (dsDNA).

During DNA replication and transcription, DNA and RNA polymerases translocate along single-stranded DNA within DNA bubbles that are generated by strand separation. As the ends of the DNA cannot rotate freely, movement of these enzymes leads to overwinding of the double-stranded DNA in front, and underwinding in the wake of the replication or transcription bubbles. The torsional stress caused by positive supercoiling ahead of these complexes inhibits further strand separation and progression of the respective process. Replication of circular covalently closed DNA leads to the generation of intertwined molecules, so-called catenanes. DNA topoisomerases remove topological stress to allow for the progression of replication and transcription, and separate catenated replication products for segregation to daughter cells.

Changes in DNA topology require cleavage and religation of the phosphodiester backbone. Strand cleavage by DNA topoisomerases is mediated by one (type I) or two (type II) catalytic tyrosines that act as nucleophiles, leading to the formation of a covalent phosphoro-tyrosyl intermediate with one or both of the cleaved strands. Type IA enzymes form a covalent bond with the 5′-end of the cleaved DNA strand, whereas type IB enzymes bind to the 3′-end. After DNA cleavage, type IA topoisomerases catalyze DNA relaxation by transporting the non-cleaved DNA strand through the gap in the cleaved strand (strand passage mechanism).

Reverse gyrase is a special topoisomerase in which a type IA topoisomerase domain cooperates with a helicase module to introduce positive supercoils into DNA in an ATP-dependent reaction. It is believed to protect the DNA of hyperthermophilic bacteria from damage at high temperatures. In contrast to type IA enzymes, type IB topoisomerases allow for controlled rotation of the free 5′-end of the DNA, leading to relaxation of positively and negatively supercoiled DNA. At the end of the supercoiling reaction, the DNA is religated.

Gyrases

Gyrases are one type of DNA topoisomerase (as the name suggests, interconverting topological isomers of DNA).

They catalyze the introduction or relaxation of DNA supercoils, as well as catenation and decatenation. Members of the type I topoisomerase family cleave a single strand of their double-stranded DNA substrate, whereas enzymes of the type II family cleave both DNA strands. Bacterial DNA gyrase, a type II topoisomerase, catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. Gyrase is not present in humans, and constitutes an attractive drug target for the treatment of bacterial and parasite infections. DNA supercoiling by gyrase is believed to occur by a strand passage mechanism, in which one segment of the double-stranded DNA substrate is passed through a (transient) break in a second segment. This mechanism requires the coordinated opening and closing of three protein interfaces, so-called gates, to ensure the directionality of strand passage toward negative supercoiling.

Single molecule fluorescence resonance energy transfer (smFRET) has allowed us to look at the precise mechanism by which gyrases work.

This relies on two separate fluorescent dye moieties (fluorophores) genetically engineered onto a protein. One of these is the “donor” and emits light in the absorption range of the “acceptor”. Based on the intensity of each signal, we can gauge the donor-acceptor distance and thus piece together the locations of the molecules' CTD and NTD with ease under different conditions (here, in the presence or absence of DNA in gyrase's grasp).

DNA gyrase consists of two GyrA- and two GyrB-subunits that form the active heterotetramer.

GyrA is subdivided into an N-terminal domain (NTD) comprised of a winged helix domain (WHD), a tower domain, and a coiled coil domain, and a C-terminal domain (CTD, Fig. 2A). GyrB consists of an N-terminal ATPase domain of the GHKL-family (for GyrB-Hsp90-histidine/serine protein kinases-MutL), a transducer domain and a C-terminal topoisomerase primase (TOPRIM) domain).

The GyrA NTD forms a heart-shaped dimer, stabilised by two gates to border the central cavity. When two GyrB subunits contact the towers (pink) and the winged helix domains (blue) with their (green) TOPRIM domains, the heterotetramer springs together forming the all-important DNA-gate.

ATP binding to the GyrB ATPase domain (not marked on the main diagram, but next to the N-gate shown hovering just over the middle of the central region) causes dimerisation and thus N-gate closure ‒ an ATP-operated clamp.

Just as a note for accuracy, the molecule studied is not ATP but ADPNP (5′-adenosine-β,γ-imidotriphosphate): a non-hydrolysable ATP analogue

The transducer domain (orange helix, partially shown) has been suggested to mediate communication between the DNA- and N-gates. The GyrA-CTD forms a six-bladed β-pinwheel structure.

This crystal structure comes from the thermophilic archaeon Thermus thermophilus — only non-overlapping fragments of more common model organisms such as E. coli are available, which aren't enough to build up a coherent (or reliable) molecular form.

As shown in the strand passage mechanism, DNA supercoiling by gyrase is proposed to occur via coordinated opening and closing of these N-, DNA- and C-gates, following initiation in which DNA wraps around gyrase in a positive node.

The gate (G)-segment, bound to the DNA-gate of gyrase, is cleaved by nucleophilic attack of the catalytic tyrosines. ATP binding and closure of the N-gate then lead to the capture of a transport (T-)segment in the upper cavity of gyrase. Opening of the DNA-gate allows the T-segment to relocate from the upper to the bottom cavity, which constitutes the name-giving strand passage step. The DNA-gate then closes, and the G-segment is religated. The T-segment leaves the bottom cavity through the C-gate. ATP hydrolysis leads to N-gate re-opening, and resetting of the enzyme allows for a subsequent catalytic cycle.

Overall, one supercoiling cycle corresponds to the passage of the transported DNA through the N-, DNA- and C-gates. In support of this mechanism, cross-linking of individual gates in gyrase abrogates the DNA supercoiling activity. A “double lock” rule for type IIA topoisomerases has been put forward by Roca, postulating that a gate can only open when the two other gates are closed. In this model, the tight temporal regulation of gate opening and closing ensures the directionality of strand passage catalyzed by type II topoisomerases. From single molecule FRET (smFRET) experiments dissecting individual conformational changes of B. subtilis DNA gyrase at the beginning of the catalytic cycle, a picture of the cascade of DNA- and nucleotide-induced conformational changes, and of the coordination of these conformational changes during the supercoiling reaction catalyzed by gyrase is now beginning to emerge.

DNA bound to the DNA-gate of topoisomerase II is bent quite strongly away from linear, and there's been suggestion that this is linked to the cleavage (less distorted DNA giving lower FRET efficiency is seen in cleavage-deficient enzymes).

Altogether, these experiments suggest that the severely distorted DNA may represent DNA that is ready to be or has already been cleaved, and places the DNA distortion at a point in the catalytic cycle close to cleavage (of the first DNA-strand). The fraction of severely distorted DNA exceeds the fraction of cleaved DNA, however, indicating that cleavage and distortion of the DNA are distinct events.

Once plasmid DNA is captured in the upper cavity of gyrase by the ATP-operated clamp at the N-gate, the DNA-gate has to then open. This has become known as the “double lock rule” — the DNA gate can only be open when the N- and C-gates are closed, reducing the risk of harmful double strand breaks.

The N-gate appears to be communicating/triggering the DNA-gate, though as yet we know not how.

A recent study has revealed an auto-inhibitory element in the topoisomerase IV CTDs that suppresses DNA relaxation, thereby favoring decatenation, although the mechanism is unclear. Altogether, ATP-dependent relaxation by topoisomerase II, negative DNA supercoiling by DNA gyrase, and DNA decatenation by topoisomerase IV are thus based on the same structural and mechanistic framework, and are achieved by different fine-tuning of a common theme.

The unknown triggers seem to be the next stage of exploration, and the authors conclude that single-molecule methods will be the way in which such subtle movements are elucidated, giving results in real-time, a detailed understanding of which may bear new, precision-engineered drug targets.

⧉  Gubaev and Klostermeier (2014) The mechanism of negative DNA supercoiling: A cascade of DNA-induced conformational changes prepares gyrase for strand passage. DNA Repair, 16: pp. 23-34

See also

⧉  DNA Topology by Maxwell and Bates (OUP, 2005)

A collaboration of Dutch chemists and microbiologists at Radboud University Nijmegen has confirmed the existence of a uniquely shaped enzyme. CS₂ hydrolase, in a form known as a catenane, consists of two (one single- and one double-ringed) interlocked macrocycles, maintained via weak non-covalent interactions in a manner never before seen in biology.

Verification by size exclusion chromatography, multi-angle laser light scattering and native mass spectrometric analyes has established that the enzyme structure, from thermophilic archaeon Acidianus A1-3 is indeed entwined.

In an earlier paper, the regulatory role this odd conformation plays in catalysis is described by microbiologist Mike Jetten, who first found the primal archaeon in the mudpots of Italian volcanic solfataras, where they had been obtaining their energy by converting CS₂ into H₂S and CO₂ using CS₂ hydrolase.

The enzyme monomer displays a typical β-carbonic anhydrase fold and active site, yet CO₂ is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical β-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme's substrate specificity for CS₂, which is hydrophobic.