Crystal Structure of an Intact Type II DNA Topoisomerase: Insights into DNA Transfer Mechanisms

Graille, Marc; Cladière, Lionel; Durand, Dominique; Lecointe, François; Gadelle, Danièle; Quevillon‐Chéruel, Sophie; Vachette, Patrice; Forterre, Patrick; Tilbeurgh, Herman van

doi:10.1016/j.str.2007.12.020

Cited by 64 publications

(81 citation statements)

References 51 publications

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“…Current evidence supports the existence of bent G segments in yeast topo II 22 and topo IV. 13 Structural data shows that topo VI lacks the tower domain and the conserved isoleucine found in topo II, both of which are shown in the DNA-bound structure of yeast topo II to facilitate bending, 24–26 consistent with the idea of a correlation between G-segment bending and topology simplification. However, as the current topo VI crystal structures lack DNA this remains to be established.…”

Section: Resultsmentioning

confidence: 66%

How Do Type II Topoisomerases Use ATP Hydrolysis to Simplify DNA Topology beyond Equilibrium? Investigating the Relaxation Reaction of Nonsupercoiling Type II Topoisomerases

Stuchinskaya

Mitchenall

Schoeffler

et al. 2009

Journal of Molecular Biology

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DNA topoisomerases control the topology of DNA (e.g. the level of supercoiling) in all cells. Type IIA topoisomerases are ATP-dependent enzymes that have been shown to simplify the topology of their DNA substrates to a level beyond that expected at equilibrium (i.e. more relaxed than the product of relaxation by ATP-independent enzymes, such as type I topoisomerases, or a lower than equilibrium level of catenation). The mechanism of this effect is currently unknown, although several models have been suggested. We have analysed the DNA relaxation reactions of type II topoisomerases to further explore this phenomenon. We find that all type IIA topoisomerases tested exhibit the effect to a similar degree and that it is not dependent on the C-terminal domains of the enzymes. As recently reported, the type IIB topoisomerase, topo VI (which is only distantly related to the type IIA enzymes), does not exhibit topology simplification. We find that topology simplification is not significantly dependent on circle size in the range ~2–9 kbp, and is not altered by reducing the free energy available from ATP hydrolysis by varying the ATP:ADP ratio. A direct test of one model (DNA tracking, i.e. sliding of a protein clamp along DNA to trap supercoils) suggests that this is unlikely to be the explanation for the effect. We conclude that geometric selection of DNA segments by the enzymes is likely to be a primary source of the effect but that it is possible that other factors contribute. We also speculate whether topology simplification might simply be an evolutionary relic, with no adaptive significance.

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Section: Resultsmentioning

confidence: 66%

How Do Type II Topoisomerases Use ATP Hydrolysis to Simplify DNA Topology beyond Equilibrium? Investigating the Relaxation Reaction of Nonsupercoiling Type II Topoisomerases

Stuchinskaya

Mitchenall

Schoeffler

et al. 2009

Journal of Molecular Biology

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“…Structures of topoisomerase VI from S. shibatae (pdb code: 2ZBK; (10)) and Methanosarcina mazei (pdb code: 2Q2E; (9)) were used as templates. Despite their low sequence similarities, the three full-length topoisomerase VIII enzymes have the same modular organization as topoisomerase VI and contain five domains.…”

Section: Resultsmentioning

confidence: 99%

DNA topoisomerase VIII: a novel subfamily of type IIB topoisomerases encoded by free or integrated plasmids in Archaea and Bacteria

et al. 2014

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Type II DNA topoisomerases are divided into two families, IIA and IIB. Types IIA and IIB enzymes share homologous B subunits encompassing the ATP-binding site, but have non-homologous A subunits catalyzing DNA cleavage. Type IIA topoisomerases are ubiquitous in Bacteria and Eukarya, whereas members of the IIB family are mostly present in Archaea and plants. Here, we report the detection of genes encoding type IIB enzymes in which the A and B subunits are fused into a single polypeptide. These proteins are encoded in several bacterial genomes, two bacterial plasmids and one archaeal plasmid. They form a monophyletic group that is very divergent from archaeal and eukaryotic type IIB enzymes (DNA topoisomerase VI). We propose to classify them into a new subfamily, denoted DNA topoisomerase VIII. Bacterial genes encoding a topoisomerase VIII are present within integrated mobile elements, most likely derived from conjugative plasmids. Purified topoisomerase VIII encoded by the plasmid pPPM1a from Paenibacillus polymyxa M1 had ATP-dependent relaxation and decatenation activities. In contrast, the enzyme encoded by mobile elements integrated into the genome of Ammonifex degensii exhibited DNA cleavage activity producing a full-length linear plasmid and that from Microscilla marina exhibited ATP-independent relaxation activity. Topoisomerases VIII, the smallest known type IIB enzymes, could be new promising models for structural and mechanistic studies.

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“…The presence of a single broad maximum in the RSV Gag distribution, as well as its smooth shape, appears to preclude the presence of a single conformer and is consistent with a multidomain protein. Bimodal distributions in PDDF plots have been observed in some proteins with rigid, unique conformations (41,42). D max , the distance r at which the p(r) drops to zero, is indicative of the longest dimension in the molecule.…”

Section: Conformation Of Gag and Gag Truncations In Solutionmentioning

confidence: 99%

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

Dick

Datta

Nanda

et al. 2015

J Virol

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Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. IMPORTANCERetroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs. Many of the principles underlying retrovirus assembly are understood. For example, the retrovirus structural protein, Gag, is able to assemble into morphologically normal virus-like particles (VLPs) in cells (1) and as a purified protein in vitro (2). The structures and functions of the multiple Gag domains are known. Thus, the MA domain mediates binding to the plasma membrane (PM), where for most retroviruses the Gag lattice is formed and budding occurs, the CA domain and immediately adjoining sequences engage in critical contacts to form the hexagonal Gag lattice, t...

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Crystal Structure of an Intact Type II DNA Topoisomerase: Insights into DNA Transfer Mechanisms

Cited by 64 publications

References 51 publications

How Do Type II Topoisomerases Use ATP Hydrolysis to Simplify DNA Topology beyond Equilibrium? Investigating the Relaxation Reaction of Nonsupercoiling Type II Topoisomerases

How Do Type II Topoisomerases Use ATP Hydrolysis to Simplify DNA Topology beyond Equilibrium? Investigating the Relaxation Reaction of Nonsupercoiling Type II Topoisomerases

DNA topoisomerase VIII: a novel subfamily of type IIB topoisomerases encoded by free or integrated plasmids in Archaea and Bacteria

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

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