Separate and combined biochemical activities of the subunits of a naturally split reverse gyrase.
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ABSTRACT:
SUBMITTER: Capp C
PROVIDER: S-EPMC3000944 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Reverse gyrase is a peculiar DNA topoisomerase, specific of thermophilic microorganisms, which induces positive supercoiling into DNA molecules in an ATP-dependent reaction. It is a modular enzyme and comprises an N-terminal helicase-like module fused to a C-terminal topoisomerase IA-like domain. The exact molecular mechanism of this unique reaction is not understood, and a fundamental mechanistic question is how its distinct steps are coordinated. We studied the cross-talk between the components of this molecular motor and probed communication between the DNA-binding sites and the different activities (DNA relaxation, ATP hydrolysis and positive supercoiling). We show that the isolated ATPase and topoisomerase domains of reverse gyrase form specific physical interactions, retain their own DNA binding and enzymatic activities, and when combined cooperate to achieve the unique ATP-dependent positive supercoiling activity. Our results indicate a mutual effect of both domains on all individual steps of the reaction. The C-terminal domain shows ATP-independent topoisomerase activity, which is repressed by the N-terminal domain in the full-length enzyme; experiments with the isolated domains showed that the C-terminal domain has stimulatory influence on the ATPase activity of the N-terminal domain. In addition, the two domains showed a striking reciprocal thermostabilization effect.
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Project description:Like all hyperthermophiles yet tested, the bacterium Thermotoga maritima contains a reverse gyrase. Here we show that it contains also a DNA gyrase. The genes top2A and top2B encoding the two subunits of a DNA gyrase-like enzyme have been cloned and sequenced. The Top2A (type II DNA topoisomerase A protein) is more similar to GyrA (DNA gyrase A protein) than to ParC [topoisomerase IV (Topo IV) C protein]. The difference is especially striking at the C-terminal domain, which differentiates DNA gyrases from Topo IV. DNA gyrase activity was detected in T. maritima and purified to homogeneity using a novobiocin-Sepharose column. This hyperhermophilic DNA gyrase has an optimal activity around 82-86 degrees C. In contrast to plasmids from hyperthermophilic archaea, which are from relaxed to positively supercoiled, we found that the plasmid pRQ7 from Thermotoga sp. RQ7 is negatively supercoiled. pRQ7 became positively supercoiled after addition of novobiocin to cell cultures, indicating that its negative supercoiling is due to the DNA gyrase of the host strain. The findings concerning DNA gyrase and negative supercoiling in Thermotogales put into question the role of reverse gyrase in hyperthermophiles.
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Project description:Reverse gyrase is a DNA topoisomerase specific for hyperthermophilic bacteria and archaea. It catalyzes the peculiar ATP-dependent DNA-positive supercoiling reaction and might be involved in the physiological adaptation to high growth temperature. Reverse gyrase comprises an N-terminal ATPase and a C-terminal topoisomerase domain, which cooperate in enzyme activity, but details of its mechanism of action are still not clear. We present here a functional characterization of PcalRG, a novel reverse gyrase from the archaeon Pyrobaculum calidifontis. PcalRG is the most robust and processive reverse gyrase known to date; it is active over a wide range of conditions, including temperature, ionic strength, and ATP concentration. Moreover, it holds a strong ATP-inhibited DNA cleavage activity. Most important, PcalRG is able to induce ATP-dependent unwinding of synthetic Holliday junctions and ATP-stimulated annealing of unconstrained single-stranded oligonucleotides. Combined DNA unwinding and annealing activities are typical of certain helicases, but until now were shown for no other reverse gyrase. Our results suggest for the first time that a reverse gyrase shares not only structural but also functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability.
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Project description: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.
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Project description:Amplicon sequencing variants (ASVs) have been proposed as an alternative to operational taxonomic units (OTUs) for analyzing microbial communities. ASVs have grown in popularity, in part because of a desire to reflect a more refined level of taxonomy since they do not cluster sequences based on a distance-based threshold. However, ASVs and the use of overly narrow thresholds to identify OTUs increase the risk of splitting a single genome into separate clusters. To assess this risk, I analyzed the intragenomic variation of 16S rRNA genes from the bacterial genomes represented in an rrn copy number database, which contained 20,427 genomes from 5,972 species. As the number of copies of the 16S rRNA gene increased in a genome, the number of ASVs also increased. There was an average of 0.58 ASVs per copy of the 16S rRNA gene for full-length 16S rRNA genes. It was necessary to use a distance threshold of 5.25% to cluster full-length ASVs from the same genome into a single OTU with 95% confidence for genomes with 7 copies of the 16S rRNA, such as Escherichia coli. This research highlights the risk of splitting a single bacterial genome into separate clusters when ASVs are used to analyze 16S rRNA gene sequence data. Although there is also a risk of clustering ASVs from different species into the same OTU when using broad distance thresholds, these risks are of less concern than artificially splitting a genome into separate ASVs and OTUs. IMPORTANCE 16S rRNA gene sequencing has engendered significant interest in studying microbial communities. There has been tension between trying to classify 16S rRNA gene sequences to increasingly lower taxonomic levels and the reality that those levels were defined using more sequence and physiological information than is available from a fragment of the 16S rRNA gene. Furthermore, the naming of bacterial taxa reflects the biases of those who name them. One motivation for the recent push to adopt ASVs in place of OTUs in microbial community analyses is to allow researchers to perform their analyses at the finest possible level that reflects species-level taxonomy. The current research is significant because it quantifies the risk of artificially splitting bacterial genomes into separate clusters. Far from providing a better representation of bacterial taxonomy and biology, the ASV approach can lead to conflicting inferences about the ecology of different ASVs from the same genome.
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Project description:Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds ?2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds ?1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque.
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Project description:Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.
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Project description:We disrupted the reverse gyrase gene from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. An apparent positive supercoiling activity that was observed in the host strain was not found in the disruptant strain. We found that a lack of reverse gyrase led to a retardation in growth that was more striking at higher temperatures. However, the disruption of the reverse gyrase gene did not lead to a lethal phenotype at 90 degrees C. This study provides experimental evidence that reverse gyrase is not a prerequisite for hyperthermophilic life.
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