ATP Hydrolysis Activity Research Articles

Inhibition of the human secretory pathway Ca2+, Mn2+–ATPase1a by 1,3-thiazole derivatives

The human Golgi/secretory pathway Ca2+,Mn2+-ATPase 1 (hSPCA1) transports Ca2+ and Mn2+ into the Golgi lumen. Studies of the biological functions of hSPCA1 are limited by a lack of selective pharmacological tools for SPCA1 inhibition. The aim of this study was therefore to identify compounds that specifically inhibit hSPCA1 activity. We found that five 1,3-thiazole derivatives exhibited inhibitory action towards the ATP hydrolysis activity of hSPCA1a in a concentration-dependent manner. Among the derivatives tested, compound 1 was the most potent, completely inhibiting hSPCA1a activity with a half-maximal inhibition (IC50) value of 0.8 μM. Compound 1 also partially inhibited the activity of another Ca2+,Mn2+-ATPase (hSPCA2) and Ca2+-ATPase (rSERCA1a), but had no effect on Na+,K+-ATPase or H+,K+-ATPase. Treatment of HeLa cells with compound 1 led to fragmentation of the Golgi ribbon into smaller stacks. In addition, compound 1 mobilized intracellular Ca2+ in HeLa cells that had been pre-treated with thapsigargin. Therefore, based on its selectivity and potency, compound 1 may be a valuable tool with which to further explore the role of SPCA1 in cellular processes.

Exploring the Role of Msh2‐Msh6 in Processing Holliday Junction DNA

Msh2‐Msh6 is a eukaryotic ATP‐dependent mismatch repair (MMR) protein that plays a key role in maintaining the fidelity of the genetic code. The protein recognizes and initiates repair for single base pair mismatches and short insertion deletion loops (IDL). Msh2‐Msh6 functions via a molecular switch mechanism, whereby ATP hydrolysis and ADP/ATP exchange facilitates the binding and release of the protein from the mismatch. Previous research has revealed that the protein binds G:T mismatch DNA with the highest affinity. Interestingly, in‐solution binding assays have revealed that Msh2‐Msh6 binds Holliday Junction DNA, a key recombination intermediate, with 1:1 stoichiometry and a similarly high affinity. However, the functional importance and structural details of this interaction are unclear. Thus far, X‐ray crystallography and Molecular Dynamics (MD) simulations are limited to the protein’s interactions with mismatch DNA only. To address the lack of structural information, we modeled the Msh2‐Msh6‐HJ interaction using the human Msh2‐Msh6 crystal structure in which we have docked in junction DNA instead of the mismatched DNA. The junction DNA used in all experiments and simulations is J3, a non‐migrating junction with 17 base pair long arms. These MD simulations will help refine the structural model and assess significant interactions between the protein and DNA. Furthermore, ATPase assays and equilibrium binding assays are used to elucidate the protein’s ATP hydrolysis activity and binding affinity in the presence of G:T mismatch DNA, J3 junction DNA, and J3 junction DNA containing a G:T mismatch. Preliminary experiments show that relative to the apoprotein’s basal ATPase activity, ATP hydrolysis is weakly stimulated when Msh2‐Msh6 is bound to G:T mismatch and J3. Gel mobility shift assays (GMSA) reveal binding affinities in the range of 9.1±2.6 nM to 23.5±11.8 nM for G:T mismatch, J3 junction, and mismatched J3 junction. These data illustrate that Msh2‐Msh6 not only binds G:T mismatch and J3 with similarly high affinity, but also that introducing a mismatch in J3 does not modulate the protein’s affinity for the junction. In addition, the GMSA data suggest that Msh2‐Msh6‐HJ binding may take on multiple conformations, consistent with previous Förster resonance energy transfer (FRET) calculations that show Msh2‐Msh6 opening the junction after binding.

Effect of Fe3+ on Na,K-ATPase: Unexpected activation of ATP hydrolysis.

Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na+ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl3 increased the intracellular storage of iron, catalase activity, the production of H2O2 and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.

What happens when the mitochondrial H+-translocating F1FO-ATP(hydrol)ase becomes a molecular target of calcium? The pore opens

The F1FO-ATPase has Mg2+ cofactor as the natural divalent cation to support the bifunctional activity of ATP synthesis and hydrolysis. Different physio(patho)logical conditions permit the molecular interaction of Ca2+ with the enzyme and the modification of the biological role. Three distinct binding regions of Ca2+ have been localized on the enzyme complex: one in the F1 catalytic sites and the other two sites in the membrane-embedded domain FO. In all likelihood, Ca2+-activated enzyme most frequently works as an H+-translocating F1FO-ATP(hydrol)ase with a monofunctional activity that triggers the formation of mitochondrial permeability transition pore (mPTP) phenomenon. The protein(s) component of the mPTP is considered an arcane mystery. However, the F1FO-ATPase could reveal the molecular mechanism of pore opening when Ca2+ is bound to the enzyme. In this regard, the role of Ca2+-dependent function of the F1FO-ATPase in the formation of the mPTP is discussed.

Modification of plasma membrane H+-ATPase in Masson pine (Pinus massoniana Lamb.) seedling roots adapting to acid deposition.

To understand the regulation of roots plasma membrane H+-ATPase in Masson pine responding to acid deposition, the changes in biomass, plant morphology, intracellular H+, enzyme activity and H+-ATPase genes expression in Masson pine seedlings exposed to simulated acid rain (SAR, pH 5.6 and 4.6) with and without vanadate were studied. Simulated acid rain exposure for 60days increased the intracellular H+ in pine roots whether added with 0.1mM Na3VO4 or not. The growth of seedlings treated with SAR was maintained well, even the primary lateral root length, root dry weight and number of root tips in seedlings exposed to SAR at pH 4.6 were higher than that of the control (pH 6.6). However, the addition of vanadate resulted in severe growth inhibition and obvious decline in morphological parameters. Similarly, ATP hydrolytic activity and H+ transport activity of roots plasma membrane H+-ATPase, both were stimulated by SAR whereas they were inhibited by vanadate, and the highest activity stimulation was observed in pine roots subjected to SAR at pH4.6. In addition, SAR also induced the expression of the investigated H+-ATPase subunits (atpB, atpE, atpF, atpH and atpI). Therefore, the roots plasma membrane H+-ATPase is instrumental in the growth of Masson pine seedlings adapting to acid rain by a manner of pumping more protons across the membrane through enhancing its activity, and which involves the upregulated gene expression of roots H+-ATPase subunits at transcriptional level.

Residues at the interface between zinc binding and winged helix domains of human RECQ1 play a significant role in DNA strand annealing activity.

RECQ1 is the shortest among the five human RecQ helicases comprising of two RecA like domains, a zinc-binding domain and a RecQ C-terminal domain containing the winged-helix (WH). Mutations or deletions on the tip of a β-hairpin located in the WH domain are known to abolish the unwinding activity. Interestingly, the same mutations on the β-hairpin of annealing incompetent RECQ1 mutant (RECQ1T1) have been reported to restore its annealing activity. In an attempt to unravel the strand annealing mechanism, we have crystallized a fragment of RECQ1 encompassing D2–Zn–WH domains harbouring mutations on the β-hairpin. From our crystal structure data and interface analysis, we have demonstrated that an α-helix located in zinc-binding domain potentially interacts with residues of WH domain, which plays a significant role in strand annealing activity. We have shown that deletion of the α-helix or mutation of specific residues on it restores strand annealing activity of annealing deficient constructs of RECQ1. Our results also demonstrate that mutations on the α-helix induce conformational changes and affects DNA stimulated ATP hydrolysis and unwinding activity of RECQ1. Our study, for the first time, provides insight into the conformational requirements of the WH domain for efficient strand annealing by human RECQ1.

The Activity of Native Vacuolar Proton-ATPase in an Oscillating Electric Field - Demystifying an Apparent Effect of Music on a Biomolecule.

The effect of an oscillating electric field generated from music on yeast vacuolar proton-ATPase (V-ATPase) activity in its native environment is reported. An oscillating electric field is generated by electrodes that are immersed into a dispersion of yeast vacuolar membrane vesicles natively hosting a high concentration of active V-ATPase. The substantial difference in the ATP hydrolysing activity of V-ATPase under the most stimulating and inhibiting music is unprecedented. Since the topic, i.e., an effect of music on biomolecules, is very attractive for non-scientific, esoteric mystification, we provide a rational explanation for the observed new phenomenon. Good correlation is found between changes in the specific activity of the enzyme and the combined intensity of certain frequency bands of the Fourier spectra of the music clips. Most prominent identified frequencies are harmonically related to each other and to the estimated rotation rate of the enzyme. These results lead to the conclusion that the oscillating electric field interferes with periodic trans-membrane charge motions in the working enzyme.

Vanadate inhibits Feo-mediated iron transport in Vibrio cholerae.

Iron is an essential element for Vibrio cholerae to survive, and Feo, the major bacterial system for ferrous iron transport, is important for growth of this pathogen in low-oxygen environments. To gain insight into its biochemical mechanism, we evaluated the effects of widely used ATPase inhibitors on the ATP hydrolysis activity of the N-terminal domain of V. cholerae FeoB. Our results showed that sodium orthovanadate and sodium azide effectively inhibit the catalytic activity of the N-terminal domain of V. cholerae FeoB. Further, sodium orthovanadate was the more effective inhibitor against V. cholerae ferrous iron transport in vivo. These results contribute to a more comprehensive biochemical understanding of Feo function, and shed light on designing effective inhibitors against bacterial FeoB proteins.

Cyperus esculentus L. and Tetracarpidium conophorum Müll. Arg. Supplemented Diet Improved Testosterone Levels, Modulated Ectonucleotidases and Adenosine Deaminase Activities in Platelets from L-NAME-Stressed Rats.

In hypertensive individuals, platelet morphology and function have been discovered to be altered, and this has been linked to the development of vascular disease, including erectile dysfunction (ED). The impact of nutritional supplementation with Cyperus esculentus (tiger nut, TN) and Tetracarpidium conophorum (walnut, WN) on androgen levels, ectonucleotidases, and adenosine deaminase (ADA) activities in platelets from L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) challenged rats were investigated. We hypothesized that these nuts may show a protective effect on platelets aggregation and possibly enhance the sex hormones, thereby reverting vasoconstriction. Wistar rats (male; 250–300 g; n = 10) were grouped into seven groups as follows: basal diet control group (I); basal diet/L-NAME/Viagra (5 mg/kg/day) as positive control group (II); ED-induced group (basal diet/L-NAME) (III); diet supplemented processed TN (20%)/L-NAME (IV); diet supplemented raw TN (20%)/L-NAME (V); diet supplemented processed WN (20%)/L-NAME (VI); and diet supplemented raw WN (20%)/L-NAME (VII). The rats were given their regular diet for 2 weeks prior to actually receiving L-NAME (40 mg/kg/day) for ten days to induce hypertension. Platelet androgen levels, ectonucleotidases, and ADA were all measured. L-NAME considerably lowers testosterone levels (54.5 ± 2.2; p < 0.05). Supplementing the TN and WN diets revealed improved testosterone levels as compared to the control (306.7 ± 5.7), but luteinizing hormone levels remained unchanged. Compared to control groups, the L-NAME-treated group showed a rise in ATP (127.5%) hydrolysis and ADA (116.7%) activity, and also a decrease in ADP (76%) and AMP (45%) hydrolysis. Both TN and WN supplemented diets resulted in substantial (p < 0.05) reversal effects. Enhanced testosterone levels and modulation of the purinergic system in platelets by TN and WN could be one of the mechanisms by which they aid in vasoconstriction control.

Functional stability of CFTR depends on tight binding of ATP at its degenerate ATP-binding site.

Opening of the cystic fibrosis transmembrane conductance regulator (CFTR) channel is coupled to the motion of its two nucleotide-binding domains: they form a heterodimer sandwiching two functionally distinct ATP-binding sites (sites 1 and 2). While active ATP hydrolysis in site 2 triggers rapid channel closure, the functional role of stable ATP binding in the catalysis-incompetent (or degenerate) site 1, a feature conserved in many other ATP-binding cassette (ABC) transporter proteins, remains elusive. Here, we found that CFTR loses its prompt responsiveness to ATP after the channel is devoid of ATP for tens to hundreds of seconds. Mutants with weakened ATP binding in site 1 and the most prevalent disease-causing mutation, F508del, are more vulnerable to ATP depletion. In contrast, strengthening ligand binding in site 1 with N6 -(2-phenylethyl)-ATP, a high-affinity ATP analogue, or abolishing ATP hydrolysis in site 2 by the mutation D1370N, helps sustain a durable function of the otherwise unstable mutant channels. Thus, tight binding of ATP in the degenerate ATP-binding site is crucial to the functional stability of CFTR. Small molecules targeting site 1 may bear therapeutic potential to overcome the membrane instability of F508del-CFTR. KEY POINTS: During evolution, many ATP-binding cassette transporters - including the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, whose dysfunction causes cystic fibrosis (CF) - lose the ability to hydrolyse ATP in one of the two ATP-binding sites. Here we show that tight ATP binding at this degenerate site in CFTR is central for maintaining the stable, robust function of normal CFTR. We also demonstrate that membrane instability of the most common CF-causing mutant, F508del-CFTR, can be rescued by strengthening ATP binding at CFTR's degenerate site. Our data thus explain an evolutionary puzzle and offer a potential therapeutic strategy for CF.

Mitochondrial Lon protease is a gatekeeper for proteins newly imported into the matrix

Human ATP-dependent Lon protease (LONP1) forms homohexameric, ring-shaped complexes. Depletion of LONP1 causes aggregation of a broad range of proteins in the mitochondrial matrix and decreases the levels of their soluble forms. The ATP hydrolysis activity, but not protease activity, of LONP1 is critical for its chaperone-like anti-aggregation activity. LONP1 forms a complex with the import machinery and an incoming protein, and protein aggregation is linked with matrix protein import. LONP1 also contributes to the degradation of imported, aberrant, unprocessed proteins using its protease activity. Taken together, our results show that LONP1 functions as a gatekeeper for specific proteins imported into the mitochondrial matrix.

The phototroph-specific β-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria

The FoF1 synthase produces ATP from ADP and inorganic phosphate. The γ subunit of FoF1 ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole FoF1 complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire FoF1 ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of FoF1 ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of FoF1 ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.

Identification of the Merkel Cell Polyomavirus Large Tumor Antigen Ubiquitin Conjugation Residue

Merkel cell polyomavirus (MCPyV) large tumor (LT) antigen is a DNA binding protein essential for viral gene transcription and genome replication. MCPyV LT interacts with multiple E3 ligases in a phosphorylation-dependent manner, limiting its own viral replication by enhancing LT protein degradation, which is a unique mechanism for MCPyV latency. Thus, identifying LT ubiquitination sites is an important step toward understanding the biological role of these virus-host interactions that can potentially result in viral oncogenesis. The ubiquitin (Ub) attachment sites in LT were predicted by using Rapid UBIquitination (RUBI), a sequence-based ubiquitination web server. Using an immunoprecipitation approach, the lysine (Lys, K) 585 residue in LT is identified as the ubiquitin conjugation site. Lysine 585 is deleted from tumor-derived truncated LTs (tLTs), resulting in stable expression of tLTs present in cancers. Substitution of lysine 585 to arginine (Arg, R) increased LT protein stability, but impaired MCPyV origin replication, due to a loss of ATP hydrolysis activity. These findings uncover a never-before-identified ubiquitination site of LT and its importance not only in the regulation of protein turnover, but also in MCPyV genome replication.

Functional implications of residues of the B' motif of geminivirus replication initiator protein in its helicase activity.

Geminivirus replication initiator protein (Rep) is a multifunctional viral protein required for replication. During the process of viral replication, Rep acts as a site- and strand-specific endonuclease, ligase, ATPase, and helicase. B' motif and β-hairpin loop of the geminivirus Rep are conserved and important for Rep-mediated helicase activity required for viral replication. To dissect the roles of various amino acid residues of the B' motif and β-hairpin loop of the geminivirus Rep helicase in its process of unwinding DNA, we investigated eight conserved residues near the ATP active site or the ssDNA contact channel. Our strategy was to mutate these residues to alanines and investigate the effects of these mutations on various biochemical activities associated with DNA unwinding. We looked into the ATP binding, ATP hydrolysis, DNA binding, and DNA unwinding activities of the wild-type and mutant Rep proteins. These investigations showed four residues (Arg279, Asp280, Tyr287, and Pro290) affecting the DNA unwinding activity. A structural model analysis confirmed the B' loop and ssDNA binding loop to be connected through a β-hairpin structure, suggesting that changes on one loop might affect the other and that these residues function by acting in concert. Viral genomes containing Rep proteins having these mutations in the B' motif did not replicate in planta. Taken together, these results indicated all four residues to be implicated in helicase activity mediated by Rep and demonstrated the significance, for viral replication, of the B' motif and β-hairpin loop of the C-terminal region of the Rep protein.

Transport of Alzheimer's associated amyloid-β catalyzed by P-glycoprotein.

P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer's disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer's associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.

The Role of Non-Catalytic Domains of Hrp3 in Nucleosome Remodeling

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.

Sequence and functional differences in the ATPase domains of CHD3 and SNF2H promise potential for selective regulability and drugability.

Chromatin remodelers use the energy of ATP hydrolysis to regulate chromatin dynamics. Their impact for development and disease requires strict enzymatic control. Here, we address the differential regulability of the ATPase domain of hSNF2H and hCHD3, exhibiting similar substrate affinities and enzymatic activities. Both enzymes are comparably strongly inhibited in their ATP hydrolysis activity by the competitive ATPase inhibitor ADP. However, the nucleosome remodeling activity of SNF2H is more strongly affected than that of CHD3. Beside ADP, also IP6 inhibits the nucleosome translocation of both enzymes to varying degrees, following a competitive inhibition mode at CHD3, but not at SNF2H. Our observations are further substantiated by mutating conserved Q- and K-residues of ATPase domain motifs. The variants still bind both substrates and exhibit a wild-type similar, basal ATP hydrolysis. Apart from three CHD3 variants, none of the variants can translocate nucleosomes, suggesting for the first time that the basal ATPase activity of CHD3 is sufficient for nucleosome remodeling. Together with the ADP data, our results propose a more efficient coupling of ATP hydrolysis and remodeling in CHD3. This aspect correlates with findings that CHD3 nucleosome translocation is visible at much lower ATP concentrations than SNF2H. We propose sequence differences between the ATPase domains of both enzymes as an explanation for the functional differences and suggest that aa interactions, including the conserved Q- and K-residues distinctly regulate ATPase-dependent functions of both proteins. Our data emphasize the benefits of remodeler ATPase domains for selective drugability and/or regulability of chromatin dynamics.

Mechanism of DNA cleavage by the endonuclease SauUSI:a major barrier to horizontal gene transfer and antibiotic resistance in Staphylococcus aureus.

Acquisition of foreign DNA by Staphylococcus aureus, including vancomycin resistance genes, is thwarted by the ATP-dependent endonuclease SauUSI. Deciphering the mechanism of action of SauUSI could unravel the reason how it singularly plays a major role in preventing horizontal gene transfer (HGT) in S. aureus. Here, we report a detailed biochemical and structural characterization of SauUSI, which reveals that in the presence of ATP, the enzyme can cleave DNA having a single or multiple target site/s. Remarkably, in the case of multiple target sites, the entire region of DNA flanked by two target sites is shred into smaller fragments by SauUSI. Crystal structure of SauUSI reveals a stable dimer held together by the nuclease domains, which are spatially arranged to hydrolyze the phosphodiester bonds of both strands of the duplex. Thus, the architecture of the dimeric SauUSI facilitates cleavage of either single-site or multi-site DNA. The structure also provides insights into the molecular basis of target recognition by SauUSI. We show that target recognition activates ATP hydrolysis by the helicase-like ATPase domain, which powers active directional movement (translocation) of SauUSI along the DNA. We propose that a pile-up of multiple translocating SauUSI molecules against a stationary SauUSI bound to a target site catalyzes random double-stranded breaks causing shredding of the DNA between two target sites. The extensive and irreparable damage of the foreign DNA by shredding makes SauUSI a potent barrier against HGT.

Single-Molecule Imaging Reveals the Mechanism Underlying Histone Loading of Schizosaccharomyces pombe AAA+ ATPase Abo1.

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1-DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.

Analysis of Nucleoside Triphosphate Hydrolysis by Middle East Respiratory Syndrome Coronavirus Helicase

The purpose of this study is to measure the nucleoside triphosphate (NTP) hydrolysis by Middle East Respiratory Syndrome coronavirus (MERS‐CoV) helicase in the presence of different types of nucleic acids (NAs) for the determination of the optimal condition of hydrolysis activity. The steady‐state NTP hydrolysis analysis demonstrated that the MERS‐CoV helicase only (and considerably) hydrolyzed the ATP in the presence of poly(U). On the contrary, only UTP was considerably hydrolyzed in the presence of poly(C). When we used the DNAs as a substrate, the single‐stranded M13 DNA considerably stimulated the ATP and UTP hydrolysis. However, the ATP hydrolysis was not detected in the presence of ds DNA. The pH‐dependent ATP hydrolysis analysis demonstrated that the helicase optimally hydrolyzed ATP at a pH value of 7.0. The ATP hydrolysis was also performed in the presence of four different kinds of divalent cations, which demonstrated that only Mg2+ ions stimulated the ATP hydrolysis activity.