Interaction with Cfd1 Increases the Kinetic Lability of FeS on the Nbp35 Scaffold

P-loop NTPases of the ApbC/Nbp35 family are involved in FeS protein maturation in nearly all organisms and are proposed to function as scaffolds for initial FeS cluster assembly. In yeast and animals, Cfd1 and Nbp35 are homologous P-loop NTPases that form a heterotetrameric complex essential for FeS protein maturation through the cytosolic FeS cluster assembly (CIA) pathway. Cfd1 is conserved in animals, fungi, and several archaeal species, but in many organisms, only Nbp35 is present, raising the question of the unique roles played by Cfd1 and Nbp35. To begin to investigate this issue, we examined Cfd1 and Nbp35 function in budding yeast. About half of each protein was detected in a heterocomplex in logarithmically growing yeast. Nbp35 readily bound ⁵⁵Fe when fed to cells, whereas ⁵⁵Fe binding by free Cfd1 could not be detected. Rapid ⁵⁵Fe binding to and release from Nbp35 was impaired by Cfd1 deficiency. A Cfd1 mutation that caused a defect in heterocomplex stability supported iron binding to Nbp35 but impaired iron release. Our results suggest a model in which Cfd1-Nbp35 interaction increases the lability of assembled FeS on the Nbp35 scaffold for transfer to target apo-FeS proteins.     

ATPase Mechanism of the 5′-3′ DNA Helicase,RecD2: EVIDENCE FOR A PRE-HYDROLYSIS CONFORMATION CHANGE*

The superfamily 1 helicase, RecD2, is a monomeric, bacterial enzyme with a role in DNA repair, but with 5′-3′ activity unlike most enzymes from this superfamily. Rate constants were determined for steps within the ATPase cycle of RecD2 in the presence of ssDNA. The fluorescent ATP analog, mantATP (2′(3′)-O-(N-methylanthraniloyl)ATP), was used throughout to provide a complete set of rate constants and determine the mechanism of the cycle for a single nucleotide species. Fluorescence stopped-flow measurements were used to determine rate constants for adenosine nucleotide binding and release, quenched-flow measurements were used for the hydrolytic cleavage step, and the fluorescent phosphate biosensor was used for phosphate release kinetics. Some rate constants could also be measured using the natural substrate, ATP, and these suggested a similar mechanism to that obtained with mantATP. The data show that a rearrangement linked to Mg²⁺ coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA. This is also shown here for the PcrA 3′-5′ helicase and so may be a general mechanism governing superfamily 1 helicases. The mechanism accounts for the tight coupling between translocation and ATPase activity.     

Interaction between Nbp35 and Cfd1 Proteins of Cytosolic Fe-S Cluster Assembly Reveals a Stable Complex Formation in Entamoeba histolytica

Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes.     

Deletion of the Proposed Iron Chaperones IscA/SufA Results in Accumulation of a Red Intermediate Cysteine Desulfurase IscS in Escherichia coli

In Escherichia coli, sulfur in iron-sulfur clusters is primarily derived from l-cysteine via the cysteine desulfurase IscS. However, the iron donor for iron-sulfur cluster assembly remains elusive. Previous studies have shown that, among the iron-sulfur cluster assembly proteins in E. coli, IscA has a unique and strong iron-binding activity and that the iron-bound IscA can efficiently provide iron for iron-sulfur cluster assembly in proteins in vitro, indicating that IscA may act as an iron chaperone for iron-sulfur cluster biogenesis. Here we report that deletion of IscA and its paralog SufA in E. coli cells results in the accumulation of a red-colored cysteine desulfurase IscS under aerobic growth conditions. Depletion of intracellular iron using a membrane-permeable iron chelator, 2,2′-dipyridyl, also leads to the accumulation of red IscS in wild-type E. coli cells, suggesting that the deletion of IscA/SufA may be emulated by depletion of intracellular iron. Purified red IscS has an absorption peak at 528 nm in addition to the peak at 395 nm of pyridoxal 5′-phosphate. When red IscS is oxidized by hydrogen peroxide, the peak at 528 nm is shifted to 510 nm, which is similar to that of alanine-quinonoid intermediate in cysteine desulfurases. Indeed, red IscS can also be produced in vitro by incubating wild-type IscS with excess l-alanine and sulfide. The results led us to propose that deletion of IscA/SufA may disrupt the iron delivery for iron-sulfur cluster biogenesis, therefore impeding sulfur delivery by IscS, and result in the accumulation of red IscS in E. coli cells.     

A bridging [4Fe-4S] cluster and nucleotide binding are essential for function of the Cfd1-Nbp35 complex as a scaffold in iron-sulfur protein maturation

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo.     

Arabidopsis cytosolic Nbp35 homodimer can assemble both [2Fe-2S] and [4Fe-4S] clusters in two distinct domains

Iron-sulfur proteins play physiologically important roles in a variety of metabolic processes in eukaryotes. In plants, iron-sulfur cluster biosynthesis is known to take place both in mitochondria and chloroplasts. However no components that mediate iron-sulfur cluster delivery in the plant cell cytosol have been identified so far. Here we report identification and characterization of a cytosolic Nbp35 homolog named AtNbp35 from Arabidopsis thaliana. AtNbp35-deficient Arabidopsis mutants were seedling lethal. Unlike the previously characterized yeast ScNbp35 which forms a heterotetramer with ScCfd1, AtNbp35 forms a homodimer in the cytosol and can harbor both [4Fe-4S] and [2Fe-2S] clusters on its amino- and carboxyl-terminal domains, respectively. Taken together, our data suggest that Nbp35 plays a pivotal role in iron-sulfur cluster assembly and delivery in the plant cell cytosol as a bifunctional molecular scaffold.     

The essential cytosolic iron-sulfur protein Nbp35 acts without Cfd1 partner in the green lineage

In photosynthetic eukaryotes assembly components of iron-sulfur (Fe-S) cofactors have been studied in plastids and mitochondria, but how cytosolic and nuclear Fe-S cluster proteins are assembled is not known. We have characterized a plant P loop NTPase with sequence similarity to Nbp35 of yeast and mammals, a protein of the cytosolic Cfd1-Nbp35 complex mediating Fe-S cluster assembly. Genome analysis revealed that NBP35 is conserved in the green lineage but that CFD1 is absent. Moreover, plant and algal NBP35 proteins lack the characteristic CXXC motif in the C terminus, thought to be required for Fe-S cluster binding. Nevertheless, chemical reconstitution and spectroscopy showed that Arabidopsis (At) NBP35 bound a [4Fe-4S] cluster in the C terminus as well as a stable [4Fe-4S] cluster in the N terminus. Holo-AtNBP35 was able to transfer an Fe-S cluster to an apoprotein in vitro. When expressed in yeast, AtNBP35 bound 55Fe dependent on the cysteine desulfurase Nfs1 and was able to partially rescue the growth of a cfd1 mutant but not of an nbp35 mutant. The AtNBP35 gene is constitutively expressed in planta, and its disruption was associated with an arrest of embryo development. These results show that despite considerable divergence from the yeast Cfd1-Nbp35 Fe-S scaffold complex, AtNBP35 has retained similar Fe-S cluster binding and transfer properties and performs an essential function.     

NSs Encoded by Groundnut Bud Necrosis Virus Is a Bifunctional Enzyme

Groundnut bud necrosis virus (GBNV), a member of genus Tospovirus in the family Bunyaviridae, infects a large number of leguminosae and solanaceae plants in India. With a view to elucidate the function of nonstructural protein, NSs encoded by the small RNA genome (S RNA), the NSs protein of GBNV- tomato (Karnataka) [1] was over-expressed in E. coli and purified by Ni-NTA chromatography. The purified rNSs protein exhibited an RNA stimulated NTPase activity. Further, this activity was metal ion dependent and was inhibited by adenosine 5′ (β, γ imido) triphosphate, an ATP analog. The rNSs could also hydrolyze dATP. Interestingly, in addition to the NTPase and dATPase activities, the rNSs exhibited ATP independent 5′ RNA/DNA phosphatase activity that was completely inhibited by AMP. The 5′ α phosphate could be removed from ssDNA, ssRNA, dsDNA and dsRNA thus confirming that rNSs has a novel 5′ α phosphatase activity. K189A mutation in the Walker motif A (GxxxxGKT) resulted in complete loss of ATPase activity, but the 5′ phosphatase activity was unaffected. On the other hand, D159A mutation in the Walker motif B (DExx) resulted in partial loss of both the activities. These results demonstrate for the first time that NSs is a bifunctional enzyme, which could participate in viral movement, replication or in suppression of the host defense mechanism.     

Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl

To determine whether the detergent-solubilized and purified vacuolar H⁺-ATPase from plants was active in H⁺ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K⁺-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K⁺ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H⁺ pumping was electrogenic. Both H⁺ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A₁, a specific vacuolar type ATPase inhibitor. The reconstituted H⁺ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO₃⁻ but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H⁺ pumping by the reconstituted ATPase in the presence of K⁺ and valinomycin. Hence, our results support the idea that the vacuolar H⁺-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl⁻ concentration. The purified and reconstituted H⁺-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H⁺ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H⁺ translocation.     

Bioenergetic Mechanism for Nisin Resistance, Induced by the Acid Tolerance Response of Listeria monocytogenes

This study examined the bioenergetics of Listeria monocytogenes, induced to an acid tolerance response (ATR). Changes in bioenergetic parameters were consistent with the increased resistance of ATR-induced (ATR⁺) cells to the antimicrobial peptide nisin. These changes may also explain the increased resistance of L. monocytogenes to other lethal factors. ATR⁺ cells had lower transmembrane pH (ΔpH) and electric potential (Δψ) than the control (ATR⁻) cells. The decreased proton motive force (PMF) of ATR⁺ cells increased their resistance to nisin, the action of which is enhanced by energized membranes. Paradoxically, the intracellular ATP levels of the PMF-depleted ATR⁺ cells were ~7-fold higher than those in ATR⁻ cells. This suggested a role for the F_oF₁ ATPase enzyme complex, which converts the energy of ATP hydrolysis to PMF. Inhibition of the F_oF₁ ATPase enzyme complex by N′-N′-1,3-dicyclohexylcarbodiimide increased ATP levels in ATR⁻ but not in ATR⁺ cells, where ATPase activity was already low. Spectrometric analyses (surface-enhanced laser desorption ionization-time of flight mass spectrometry) suggested that in ATR⁺ listeriae, the downregulation of the proton-translocating c subunit of the F_oF₁ ATPase was responsible for the decreased ATPase activity, thereby sparing vital ATP. These data suggest that regulation of F_oF₁ ATPase plays an important role in the acid tolerance response of L. monocytogenes and in its induced resistance to nisin.     

Functional Dynamics Revealed by the Structure of the SufBCD Complex,a Novel ATP-binding Cassette (ABC) Protein That Serves as a Scaffold for Iron-Sulfur Cluster Biogenesis

ATP-binding cassette (ABC)-type ATPases are chemomechanical engines involved in diverse biological pathways. Recent genomic information reveals that ABC ATPase domains/subunits act not only in ABC transporters and structural maintenance of chromosome proteins, but also in iron-sulfur (Fe-S) cluster biogenesis. A novel type of ABC protein, the SufBCD complex, functions in the biosynthesis of nascent Fe-S clusters in almost all Eubacteria and Archaea, as well as eukaryotic chloroplasts. In this study, we determined the first crystal structure of the Escherichia coli SufBCD complex, which exhibits the common architecture of ABC proteins: two ABC ATPase components (SufC) with function-specific components (SufB-SufD protomers). Biochemical and physiological analyses based on this structure provided critical insights into Fe-S cluster assembly and revealed a dynamic conformational change driven by ABC ATPase activity. We propose a molecular mechanism for the biogenesis of the Fe-S cluster in the SufBCD complex.     

DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis

The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R₂N₂ stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6–7 nucleotides) downstream of the asymmetric recognition sequence 5′-GCCGC-3′. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage.     

Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIβ. We show that topo IIβ is a DNA-dependent ATPase that appears to fit Michaelis–Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k_cat for ATP hydrolysis by human DNA topo IIβ in the presence of DNA is 2.25 s^–1. We have characterised a topo IIβ derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the k_cat for ATP hydrolysis by 7-fold, to 0.32 s^–1, while not significantly altering the apparent K_m. The specificity constant for the interaction between ATP and topo IIβ (k_cat/K_mapp) showed a 90% reduction for βS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIβ. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.     

Reinterpreting the Action of ATP Analogs on KATP Channels

Neuroendocrine-type K_ATP channels, (SUR1/Kir6.2)₄, couple the transmembrane flux of K⁺, and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5′-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR1_E1507Q, with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5′-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[³²P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K_ATP channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.     

Werner syndrome exonuclease catalyzes structure-dependent degradation of DNA

Werner syndrome (WS) is an autosomal recessive disease characterized by early onset of many features of aging, by an unusual spectrum of cancers, and by genomic instability. The WS protein (WRN) possesses 3′→5′ DNA helicase and associated ATPase activities, as well as 3′→5′ DNA exonuclease activity. Currently, WRN is the only member of the widely distributed RecQ DNA helicase family with documented exonuclease activity. It is not known whether deficiency of the exonuclease or helicase/ATPase activities of WRN, or all of them, is responsible for various elements of the WS phenotype. WRN exonuclease has limited homology to Escherichia coli RNaseD, a tRNA processing enzyme. We show here that WRN preferentially degrades synthetic DNA substrates containing alternate secondary structures, with an exonucleolytic mode of action suggestive of RNaseD. We present evidence that structure-dependent binding of WRN to DNA requires ATP binding, while DNA degradation requires ATP hydrolysis. Apparently, the exonuclease and ATPase act in concert to catalyze structure-dependent DNA degradation. We propose that WRN protein functions as a DNA processing enzyme in resolving aberrant DNA structures via both exonuclease and helicase activities.     

Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP

The 2′,3′-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H⁺-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH₄. The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K_i of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s⁻¹. The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [³H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.     

A novel nuclease-ATPase (Nar71) from archaea is part of a proposed thermophilic DNA repair system

We have identified a novel structure-specific nuclease in highly fractionated extracts of the thermophilic archaeon Methanothermobacter thermautotrophicus (Mth). The 71 kDa protein product of open reading frame mth1090 is a nuclease with ATPase activity, which we call Nar71 (Nuclease-ATPase in Repair, 71 kDa). The nar71 gene is located in a gene neighbourhood proposed by genomics to encode a novel DNA repair system conserved in thermophiles. The biochemical characterization of Nar71 presented here is the first analysis from within this neighbourhood, and it supports the insight from genomics. Nuclease activity of Nar71 is specific for 3′ flaps and flayed duplexes, targeting single-stranded DNA (ssDNA) regions. This activity requires Mg²⁺ or Mn²⁺ and is greatly reduced in ATP. In ATP, Nar71 displaces ssDNA, also with high specificity for 3′ flap and flayed duplex DNA. Strand displacement is weak compared with nuclease activity, but in ATPγS it is abolished, suggesting that Nar71 couples ATP hydrolysis to DNA strand separation. ATPase assays confirmed that Nar71 is stimulated by ssDNA, though not double-stranded DNA. Mutation of Lys-117 in Nar71 abolished ATPase and nuclease activity, and we describe a separation-of-function mutant (K68A) that has lost ATPase activity but retains nuclease activity. A model of possible Nar71 function in DNA repair is presented.     

An essential DnaB helicase of Bacillus anthracis: identification, characterization, and mechanism of action

We have described a novel essential replicative DNA helicase from Bacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB helicase (DnaB_BA) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaB_BA displayed distinct enzymatic and kinetic properties. DnaB_BA has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5′→3′ DNA helicase activity. The stimulation of ATPase activity appeared to be a function of the length of the ssDNA template rather than of ssDNA binding alone. The highest specific activity was observed with M13mp19 ssDNA. The results presented here indicated that the ATPase activity of DnaB_BA was coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaB_BA demonstrated a strong DNA helicase activity that required ATP or dATP. Therefore, DnaB_BA has an attenuated ATPase activity and a highly active DNA helicase activity. Based on the ratio of DNA helicase and ATPase activities, DnaB_BA is highly efficient in DNA unwinding and its coupling to ATP consumption.     

Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45°C, and an optimal divalent-cation concentration of 5 mM MgCl₂. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The K_m values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 μM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3′-to-5′ direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5′ or 3′ tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3′ tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.