The helicase-like domain from "Thermotoga maritima" reverse gyrase : catalytic cycle and contribution to DNA supercoiling

Reverse gyrases are the only topoisomerases capable of introducing positive supercoils into circular DNA. Their exclusive presence in thermophilic and hyperthermophilic organisms indicates a DNA thermoprotective role in vivo. In spite of the efforts to improve our knowledge of reverse gyrase, modest progress has been made since its discovery. Currently, only one crystal structure of the enzyme is available, and the most widely accepted reaction mechanism is a hypothetical one, mostly derived from the functions of enzymes related to reverse gyrase domains. In the present work we address mechanistic aspects of the reaction by exploiting the capabilities of a wide range of techniques, to elucidate the role of one module of T. maritima reverse gyrase. Reverse gyrase consists of an N-terminal helicase-like domain, fused to a C-terminal topoisomerase domain. We selected the helicase-like domain as a model of study due to its capacity to couple ATP binding and hydrolysis to DNA processing. Exploiting of these features by reverse gyrase turns this region into a key player at virtually every step of DNA supercoiling. Steady-state ATPase assays and equilibrium binding titrations with the helicase-like domain and the full-length enzyme, enabled us to prove for the first time a harnessing effect of the topoisomerase over the helicase-like domain. We showed that properties intrinsic to the helicase-like domain, like DNA-stimulated ATP hydrolysis, nucleotide-dependent affinity switch for DNA, and thermodynamic coupling between DNA binding and ATP binding and hydrolysis, are strongly reduced in the context of reverse gyrase. At that time apparent contradictions arose, from reports stating that the isolated helicase-like domain is less active than within the context of the full-length enzyme. We reconciled these differences by demonstrating that the presence of the putative N-terminal Zn-finger in the helicase-like domain construct is the cause for the decreased activity. Furthermore, we have elucidated the thermodynamic and conformational cycle of the helicase-like domain, and predicted the stages fulfilling the requirements for interdomain communication, local duplex DNA unwinding, and the stages where DNA is in a suitable state to support the supercoiling reaction. Finally, besides the use of smFRET as a tool to investigate conformational changes in solution, we have also provided high-resolution snapshots of the helicase-like domain via X-ray crystallography. We have provided the most detailed structures of this region to this date, in the apo and ADP-bound forms. They also revealed high flexibility of the linker joining the RecA domains with relative orientations far from random, and local differences in secondary structure motifs that discard the assumption of all reverse gyrases having a “monolithic” build-up. We also created a deletion mutant of the latch, region with a sui generis location, perfectly suited for interdomain communication. Previous reports stated that its deletion from reverse gyrase abolishes positive supercoiling. We demonstrated its strong involvement in DNA binding, DNA-stimulated ATP hydrolysis, and thermodynamic coupling between these processes in the isolated helicase-like domain. We also revealed its role in presenting the ssDNA to the topoisomerase domain and in guiding the strand passage and resealing, ensuring the directionality leading to the introduction of positive supercoils. Additionally, we also elucidated the nucleotide cycle and conformational transitions for this helicase-like domain mutant, which gave the first indications of why no positive supercoiling can be performed by the full-length reverse gyrase lacking the latch, and only DNA relaxation is allowed. Finally, our pre steady-state kinetic studies allowed us to fully describe the unstimulated ATPase activity of the isolated helicase-like domain. We also demonstrated for the first time its DNA unwinding activity, shedding light on the rarely documented local B-DNA duplex destabilization of helicase-like modules, appended to bigger enzymes. Additionally, the sequence of ssDNA strand release, and identification of secondary structure motifs involved in ssDNA binding at different stages were determined. Together with the finding of new conformational states via smFRET, and “targeted” supercoiling assays with the full-length enzyme, we end up proposing a detailed catalytic mechanism, similar to the one derived from the reverse gyrase structure, only this time based on and supported by a combination of kinetic, thermodynamic, and structural data

The latch modulates nucleotide and DNA binding to the helicase-like domain of Thermotoga maritima reverse gyrase and is required for positive DNA supercoiling

Reverse gyrase is the only topoisomerase that can introduce positive supercoils into DNA in an ATP-dependent process. It has a modular structure and harnesses a helicase-like domain to support a topoisomerase activity, thereby creating the unique function of positive DNA supercoiling. The isolated topoisomerase domain can relax negatively supercoiled DNA, an activity that is suppressed in reverse gyrase. The isolated helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain. Inter-domain communication thus appears central for the functional cooperation of the two domains. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the influence of the latch on nucleotide and DNA binding to the helicase-like domain, and on DNA supercoiling by reverse gyrase. We find that the latch is required for positive DNA supercoiling. It is crucial for the cooperativity of DNA and nucleotide binding to the helicase-like domain. The latch contributes to DNA binding, and affects the preference of reverse gyrase for ssDNA. Thus, the latch coordinates the individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domai

Crystal structures of Thermotoga maritima reverse gyrase: inferences for the mechanism of positive DNA supercoiling

Reverse gyrase is an ATP-dependent topoisomerase that is unique to hyperthermophilic archaea and eubacteria. The only reverse gyrase structure determined to date has revealed the arrangement of the N-terminal helicase domain and the C-terminal topoisomerase domain that intimately cooperate to generate the unique function of positive DNA supercoiling. Although the structure has elicited hypotheses as to how supercoiling may be achieved, it lacks structural elements important for supercoiling and the molecular mechanism of positive supercoiling is still not clear. We present five structures of authentic Thermotoga maritima reverse gyrase that reveal a first view of two interacting zinc fingers that are crucial for positive DNA supercoiling. The so-called latch domain, which connects the helicase and the topoisomerase domains is required for their functional cooperation and presents a novel fold. Structural comparison defines mobile regions in parts of the helicase domain, including a helical insert and the latch that are likely important for DNA binding during catalysis. We show that the latch, the helical insert and the zinc fingers contribute to the binding of DNA to reverse gyrase and are uniquely placed within the reverse gyrase structure to bind and guide DNA during strand passage. A possible mechanism for positive supercoiling by reverse gyrases is presente

The latch modulates nucleotide and DNA binding to the helicase-like domain of Thermotoga maritima reverse gyrase and is required for positive DNA supercoiling

Reverse gyrase is the only topoisomerase that can introduce positive supercoils into DNA in an ATP-dependent process. It has a modular structure and harnesses a helicase-like domain to support a topoisomerase activity, thereby creating the unique function of positive DNA supercoiling. The isolated topoisomerase domain can relax negatively supercoiled DNA, an activity that is suppressed in reverse gyrase. The isolated helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain. Inter-domain communication thus appears central for the functional cooperation of the two domains. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the influence of the latch on nucleotide and DNA binding to the helicase-like domain, and on DNA supercoiling by reverse gyrase. We find that the latch is required for positive DNA supercoiling. It is crucial for the cooperativity of DNA and nucleotide binding to the helicase-like domain. The latch contributes to DNA binding, and affects the preference of reverse gyrase for ssDNA. Thus, the latch coordinates the individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domain

The reverse gyrase helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain

Reverse gyrase is a topoisomerase that introduces positive supercoils into DNA in an ATP-dependent manner. It is unique to hyperthermophilic archaea and eubacteria, and has been proposed to protect their DNA from damage at high temperatures. Cooperation between its N-terminal helicase-like and the C-terminal topoisomerase domain is required for positive supercoiling, but the precise role of the helicase-like domain is currently unknown. Here, the characterization of the isolated helicase-like domain from Thermotoga maritima reverse gyrase is presented. We show that the helicase-like domain contains all determinants for nucleotide binding and ATP hydrolysis. Its intrinsic ATP hydrolysis is significantly stimulated by ssDNA, dsDNA and plasmid DNA. During the nucleotide cycle, the helicase-like domain switches between high- and low-affinity states for dsDNA, while its affinity for ssDNA in the ATP and ADP states is similar. In the context of reverse gyrase, the differences in DNA affinities of the nucleotide states are smaller, and the DNA-stimulated ATPase activity is strongly reduced. This inhibitory effect of the topoisomerase domain decelerates the progression of reverse gyrase through the nucleotide cycle, possibly providing optimal coordination of ATP hydrolysis with the complex reaction of DNA supercoiling

Identification of a Natural Viral RNA Motif That Optimizes Sensing of Viral RNA by RIG-I

ABSTRACT Stimulation of the antiviral response depends on the sensing of viral pathogen-associated molecular patterns (PAMPs) by specialized cellular proteins. During infection with RNA viruses, 5′-di- or -triphosphates accompanying specific single or double-stranded RNA motifs trigger signaling of intracellular RIG-I-like receptors (RLRs) and initiate the antiviral response. Although these molecular signatures are present during the replication of many viruses, it is unknown whether they are sufficient for strong activation of RLRs during infection. Immunostimulatory defective viral genomes (iDVGs) from Sendai virus (SeV) are among the most potent natural viral triggers of antiviral immunity. Here we describe an RNA motif (DVG70-114) that is essential for the potent immunostimulatory activity of 5′-triphosphate-containing SeV iDVGs. DVG70-114 enhances viral sensing by the host cell independently of the long stretches of complementary RNA flanking the iDVGs, and it retains its stimulatory potential when transferred to otherwise inert viral RNA. In vitro analysis showed that DVG70-114 augments the binding of RIG-I to viral RNA and promotes enhanced RIG-I polymerization, thereby facilitating the onset of the antiviral response. Together, our results define a new natural viral PAMP enhancer motif that promotes viral recognition by RLRs and confers potent immunostimulatory activity to viral RNA

Analysis of gene expression data from Massive Parallel Sequencing identifies so far uncharacterised regulators for meiosis with one candidate being fundamental for prophase I in male and female meiosis

Meiosis is a specialized division of germ cells in sexually reproducing organisms, which is a fundamental process with key implications for evolution and biodiversity. In two consecutive rounds of cell division, meiosis I and meiosis II, a normal, diploid set of chromosome is halved. From diploid mother cells haploid gametes are generated to create genetic individual cells. This genetic uniqueness is obtained during prophase of meiosis I by essential meiotic processes in meiotic recombination, as double strand break (DSB) formation and repair, formation of crossovers (CO) and holiday junctions (HJs). Checkpoint mechanisms ensure a smooth progress of these events. Despite extensive research key mechanisms are still not understood. Based on an analysis of Massive Parallel Sequencing (MPS) data I could identify 2 genes, Mcmdc2 and Prr19, with high implication in meiotic recombination. In the absence of Mcmdc2 both sexes are infertile and meiocytes arrest at a stage equivalent to mid-­‐pachytene in wt. Investigations of the synaptonemal complex (SC) formation revealed severe defects suggesting a role for MCMDC2 in homology search. Moreover, MCMDC2 does not seem to be essential for DSB repair, as DSB markers of early and mid recombination nodules, like DMC1 and RPA, are decreased in oocytes. Nevertheless, late recombination nodules, which are positive for MutL homolog 1 (MLH1), do not form in both sexes. The absence of the asynapsis surveillance checkpoint mechanism in Hormad2 deficient ovaries with Mcmdc2 mutant background allowed survival of oocytes. This points into the direction that Mcmdc2 knock­out oocytes get eliminated after prophase I due to failed homologous synapsis. Interestingly, MCMDC2 contains a conserved helicase domain, like the MCM protein family members MCM8 and MCM9. I therefore hyphothesize that Mcmdc2 promotes homolgy search