Reverse Gyrase, the Two Domains Intimately Cooperate to Promote Positive Supercoiling

Déclais, Anne-Cécile; Marsault, Janine; Confalonieri, Fabrice; Tour, Claire Bouthier de la; Duguet, Michel

doi:10.1074/jbc.m910091199

Cited by 65 publications

(96 citation statements)

References 27 publications

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“…The topoisomerase domain expressed on its own does weakly relax DNA in the absence of ATP (Fig. 5D), as reported for the topoisomerase domain of S. acidocaldarius reverse gyrase (21). These results indicate that the N-terminal domain represses the activity of the topoisomerase domain in the absence of nucleotide.…”

Section: Dna Binding Behavior-supporting

confidence: 67%

“…A contrasting explanation for the unidirectionality is provided by the "domain model" (6). Reverse gyrase is known to unwind DNA locally upon binding (9,21), and the domain model stipulates that the enzyme can isolate this unwound DNA topologically from the rest of the substrate. In DNA that is topologically constrained as a whole, such as a plasmid, the creation of unwound DNA at the protein binding site creates compensatory overwinding elsewhere.…”

Section: Fig 8 Topoisomerization Activity Of Reverse Gyrase Mutantmentioning

confidence: 99%

“…Then an excess of inactive reverse gyrase mutant was added to the reaction, and the 80°C incubation resumed. The inactive mutant carries a Phe in place of the catalytic Tyr-809, reducing DNA cleavage to very low levels (data not shown), without affecting the enzyme's ability to bind and unwind DNA (21). The inactive reverse gyrase should bind to the partially relaxed plasmid and create overwinding elsewhere, such that the active reverse gyrase would rebind, on average, to overwound regions.…”

Section: Fig 8 Topoisomerization Activity Of Reverse Gyrase Mutantmentioning

confidence: 99%

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Studies of a Positive Supercoiling Machine

Rodríguez

2002

Journal of Biological Chemistry

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Reverse gyrase, the only topoisomerase known to positively supercoil DNA, has an N-terminal ATPase domain that drives the activity of a topoisomerase domain. This study shows that the N-terminal domain represses topoisomerase activity in the absence of nucleotide, and nucleotide binding is sufficient to relieve the repression. A "latch" region in the N-terminal part was observed to close over the topoisomerase domain in the reverse gyrase crystal structure. Mutants lacking all or part of the latch relax DNA in the absence of nucleotide, indicating that this region mediates topoisomerase repression. The mutants also show altered DNA-dependent ATPase activity, suggesting that the latch may be involved in coupling nucleotide hydrolysis to supercoiling. It is not required for this process, however, because the mutants can still positively supercoil DNA. Nucleotide hydrolysis is essential to the specificity of reverse gyrase for increasing the linking number of DNA. Although with ATP the enzyme performs strand passage always toward increasing linking number, it can increase or decrease the linking number in the presence of a nonhydrolyzable ATP analog. This suggests that the mechanism of reverse gyrase is best described by a combination of recently proposed models.Topoisomerases participate in practically every DNA transaction, including transcription, replication, recombination, and chromosomal segregation (1). They change the topological state of DNA by altering its linking number, i.e. the number of times that one strand of the double helix crosses the other. They use a three-step mechanism of cleavage, strand passage, and religation. In the first step, a nucleophilic tyrosine attacks the phosphodiester backbone, cleaving the DNA and leading to a covalent intermediate of protein-DNA termed the "cleavage complex." Type I topoisomerases cleave one strand of the duplex; type II enzymes cleave both strands. The enzyme, covalently attached to the cut DNA, separates the free ends of the cleaved strand(s) and allows the other strand of the duplex (type I), or another region of duplex (type II), to pass through this gap. The protein then reseals the backbone of the cleaved DNA and releases the product.Although all topoisomerases can relax supercoiled DNA, only prokaryotic gyrase is able to introduce negative supercoils in DNA. This type II topoisomerase uses the energy of ATP hydrolysis to reduce the linking number, leading to an underwound, or negatively supercoiled, product. Until recently, gyrase was the only topoisomerase known to supercoil DNA. Later, another supercoiling enzyme was discovered and named reverse gyrase, because it increases the linking number, leading to overwound, or positively supercoiled, product (2, 3). Reverse gyrase is the only topoisomerase known to overwind DNA. So far it has been found exclusively in hyperthermophiles, organisms that live above ϳ80°C (4, 5). The role of the protein in vivo remains unclear (6); one suggestion is that it rewinds the DNA strands in regions of the chromosome that...

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Section: Dna Binding Behavior-supporting

confidence: 67%

Section: Fig 8 Topoisomerization Activity Of Reverse Gyrase Mutantmentioning

confidence: 99%

Section: Fig 8 Topoisomerization Activity Of Reverse Gyrase Mutantmentioning

confidence: 99%

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Studies of a Positive Supercoiling Machine

Rodríguez

2002

Journal of Biological Chemistry

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“…Reverse gyrase, an enzyme first discovered in the hyperthermophilic archaeon Sulfolobus acidocaldarius, is formed by the combination of an N-terminal helicase module and a C-terminal topoisomerase module (see Declais et al 2000 for a review). Comparative genomic analysis performed 4 years ago showed that reverse gyrase was at that time the only protein specific for hyperthermophiles (i.e., present in all hyperthermophiles and absent in all non-hyperthermophiles) (Forterre 2002).…”

Section: Introductionmentioning

confidence: 99%

Widespread distribution of archaeal reverse gyrase in thermophilic bacteria suggests a complex history of vertical inheritance and lateral gene transfers

Brochier-Armanet

Forterre

2006

Archaea

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SummaryReverse gyrase, an enzyme of uncertain funtion, is present in all hyperthermophilic archaea and bacteria. Previous phylogenetic studies have suggested that the gene for reverse gyrase has an archaeal origin and was transferred laterally (LGT) to the ancestors of the two bacterial hyperthermophilic phyla, Thermotogales and Aquificales. Here, we performed an in-depth analysis of the evolutionary history of reverse gyrase in light of genomic progress. We found genes coding for reverse gyrase in the genomes of several thermophilic bacteria that belong to phyla other than Aquificales and Thermotogales. Several of these bacteria are not, strictly speaking, hyperthermophiles because their reported optimal growth temperatures are below 80°C. Furthermore, we detected a reverse gyrase gene in the sequence of the large plasmid of Thermus thermophilus strain HB8, suggesting a possible mechanism of transfer to the T. thermophilus strain HB8 involving plasmids and transposases. The archaeal part of the reverse gyrase tree is congruent with recent phylogenies of the archaeal domain based on ribosomal proteins or RNA polymerase subunits. Although poorly resolved, the complete reverse gyrase phylogeny suggests an ancient acquisition of the gene by bacteria via one or two LGT events, followed by its secondary distribution by LGT within bacteria. Finally, several genes of archaeal origin located in proximity to the reverse gyrase gene in bacterial genomes have bacterial homologues mostly in thermophiles or hyperthermophiles, raising the possibility that they were co-transferred with the reverse gyrase gene. Our new analysis of the reverse gyrase history strengthens the hypothesis that the acquisition of reverse gyrase may have been a crucial evolutionary step in the adaptation of bacteria to high-temperature environments. However, it also questions the role of this enzyme in thermophilic bacteria and the selective advantage its presence could provide.

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“…Reverse gyrase is found in hyperthermophilic archaea and eubacteria, and the positive supercoiling activity requires a temperature above 70°C (8,9). The enzyme is a 130-kDa single polypeptide, a fusion complex of two domains (10): The carboxyl terminal half is related to topo IA, whereas the amino terminal half has an ATP-binding site and is akin to helicase, although neither the whole enzyme nor the isolated helicase-like domain shows genuine helicase activity (11). A crystal structure of the full-length reverse gyrase (12) and more recent structures with additional features (13) all support the basic two-domain construct.…”

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confidence: 99%

Direct observation of DNA overwinding by reverse gyrase

Ogawa

Yogo

Furuike

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70°C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71°C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site. A single reverse gyrase molecule processively winds the DNA for >100 turns. Bound enzyme shows moderate temperature dependence, retaining significant activity down to 50°C. The unloaded reaction rate at 71°C exceeds five turns per second, which is >10 2 -fold higher than hitherto indicated but lower than the measured ATPase rate of 20 s −1 , indicating loose coupling. The overwinding reaction sharply slows down as the torsional stress accumulates in DNA and ceases at stress of mere ∼5 pN·nm, where one more turn would cost only sixfold the thermal energy. The enzyme would thus keep DNA in a slightly overwound state to protect, but not overprotect, the genome of hyperthermophiles against thermal melting. Overwinding activity is also highly sensitive to DNA tension, with an effective interaction length exceeding the size of reverse gyrase, implying requirement for slack DNA. All results point to the mechanism where strand passage relying on thermal motions, as in topoisomerase IA, is actively but loosely biased toward overwinding.reverse gyrase | topoisomerase | magnetic tweezers | DNA overwinding | torsion R everse gyrase, discovered in 1984 in a hyperthermophilic archaeon Sulfolobus (1) which was later classified as Sulfolobus tokodaii (2), is a unique DNA topoisomerase that can introduce positive supercoils into DNA (3-7). The only other enzyme that has the gyration activity is DNA gyrase, which introduces negative supercoils. Although DNA gyrase belongs to type II topoisomerase, which changes the linking number (Lk) of dsDNA by two by cutting and religating both strands simultaneously, reverse gyrase is of type IA topoisomerase (topo IA), where one strand is cut to allow the passage of the other, resulting in the Lk changes in steps of one (3-7). Reverse gyrase is found in hyperthermophilic archaea and eubacteria, and the positive supercoiling activity requires a temperature above 70°C (8, 9). The enzyme is a 130-kDa single polypeptide, a fusion complex of two domains (10): The carboxyl terminal half is related to topo IA, whereas the amino terminal half has an ATP-binding site and is akin to helicase, although neither the whole enzyme nor the isolated helicase-like domain shows genuine helicase activity (11). A crystal structure of the full-length reverse gyrase (12) and more recent structures with additional features (13) all support the basic two-domain construct. The physiological roles of reverse gyrase are not yet fully clear, although positive supercoiling is expected to protect DNA from denaturation at the growth temperatures of hyperthermophiles.Reactions of reverse gyra...

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Reverse Gyrase, the Two Domains Intimately Cooperate to Promote Positive Supercoiling

Cited by 65 publications

References 27 publications

Studies of a Positive Supercoiling Machine

Studies of a Positive Supercoiling Machine

Widespread distribution of archaeal reverse gyrase in thermophilic bacteria suggests a complex history of vertical inheritance and lateral gene transfers

Direct observation of DNA overwinding by reverse gyrase

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