Bacillus subtilis RecN binds and protects 3'-single-stranded DNA extensions in the presence of ATP

Bacillus subtilis RecN appears to be an early detector of breaks in double-stranded DNA. In vivo, RecN forms discrete nucleoid-associated structures and in vitro exhibits Mg²⁺-dependent single-stranded (ss) DNA binding and ssDNA-dependent ATPase activities. In the presence of ATP or ADP, RecN assembles to form large networks with ssDNA molecules (designated complexes CII and CIII) that involve ATP binding and requires a 3′-OH at the end of ssDNA molecule. Addition of dATP–RecA complexes dissociates RecN from these networks, but this is not observed following addition of an ssDNA binding protein. Apparently, ATP modulates the RecN–ssDNA complex for binding to ssDNA extensions and, in vivo, RecN–ATP bound to 3′-ssDNA might sequester ssDNA ends within complexes that protect the ssDNA while the RecA accessory proteins recruit RecA. With the association of RecA to ssDNA, RecN would dissociate from the DNA end facilitating the subsequent steps in DNA repair.     

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.     

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.     

Mycobacterium tuberculosis RecA intein,a LAGLIDADG homing endonuclease,displays Mn(2+) and DNA-dependent ATPase activity

Mycobacterium tuberculosis RecA intein (PI-MtuI), a LAGLIDADG homing endonuclease, displays dual target specificity in response to alternative cofactors. While both ATP and Mn²⁺ were required for optimal cleavage of an inteinless recA allele (hereafter referred to as cognate DNA), Mg²⁺ alone was sufficient for cleavage of ectopic DNA sites. In this study, we have explored the ability of PI-MtuI to catalyze ATP hydrolysis in the presence of alternative metal ion cofactors and DNA substrates. Our results indicate that PI-MtuI displays maximum ATPase activity in the presence of cognate but not ectopic DNA. Kinetic analysis revealed that Mn²⁺ was able to stimulate PI-MtuI catalyzed ATP hydrolysis, whereas Mg²⁺ failed to do so. Using UV crosslinking, limited proteolysis and amino acid sequence analysis, we show that ³²P-labeled ATP was bound to a 14 kDa peptide containing the putative Walker A motif. Furthermore, the limited proteolysis approach disclosed that cognate DNA was able to induce structural changes in PI-MtuI. Mutation of the presumptive metal ion-binding ligands (Asp122 and Asp222) in the LAGLIDADG motifs of PI-MtuI impaired its affinity for ATP, thus resulting in a reduction in or loss of its endonuclease activity. Together, these results suggest that PI-MtuI is a (cognate) DNA- and Mn²⁺-dependent ATPase, unique from the LAGLIDADG family of homing endonucleases, and implies a possible role for ATP hydrolysis in the recognition and/or cleavage of homing site DNA sequence.     

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.     

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.     

Biochemical analysis of human Dna2

Yeast Dna2 helicase/nuclease is essential for DNA replication and assists FEN1 nuclease in processing a subset of Okazaki fragments that have long single-stranded 5′ flaps. It is also involved in the maintenance of telomeres. DNA2 is a gene conserved in eukaryotes, and a putative human ortholog of yeast DNA2 (ScDNA2) has been identified. Little is known about the role of human DNA2 (hDNA2), although complementation experiments have shown that it can function in yeast to replace ScDNA2. We have now characterized the biochemical properties of hDna2. Recombinant hDna2 has single-stranded DNA-dependent ATPase and DNA helicase activity. It also has 5′–3′ nuclease activity with preference for single-stranded 5′ flaps adjacent to a duplex DNA region. The nuclease activity is stimulated by RPA and suppressed by steric hindrance at the 5′ end. Moreover, hDna2 shows strong 3′–5′ nuclease activity. This activity cleaves single-stranded DNA in a fork structure and, like the 5′–3′ activity, is suppressed by steric hindrance at the 3′-end, suggesting that the 3′–5′ nuclease requires a 3′ single-stranded end for activation. These biochemical specificities are very similar to those of the ScDna2 protein, but suggest that the 3′–5′ nuclease activity may be more important than previously thought.     

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines

Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k_cat 415 s⁻¹), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3′ → 5′ translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the “leading” AdnB and “lagging” AdnA motor domains track in tandem, 3′ to 5′, along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5′ and 3′ terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5′ strand, while the AdnB nuclease cleaves the displaced 3′ strand. We present evidence that the distinctive “molecular ruler” function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5′-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.     

Specific inhibition of the E.coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxyribonucleotides

RecBCD is an ATP-dependent helicase and exonuclease which generates 3′ single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. The exonuclease activity is altered when RecBCD encounters a Chi sequence (5′-GCTGGTGG-3′) in double-stranded DNA (ds DNA), an event critical to the generation of the 3′-ssDNA. This study tests the effect of ssDNA oligonucleotides having a Chi sequence (Chi⁺) or a single base change that abolishes the Chi sequence (Chi^o), on the enzymatic activities of RecBCD. Our results show that a 14 and a 20mer with Chi⁺ in the center of the molecule inhibit the exonuclease and helicase activities of RecBCD to a greater extent than the corresponding Chi^o oligonucleotides. Oligonucleotides with the Chi sequence at one end, or the Chi sequence alone in an 8mer, failed to show Chi-specific inhibition of RecBCD. Thus, Chi recognition requires that Chi be flanked by DNA at either end. Further experiments indicated that the oligonucleotides inhibit RecBCD from binding to its dsDNA substrate. These results suggest that a specific site for Chi recognition exists on RecBCD, which binds Chi with greater affinity than a non-Chi sequence and is probably adjacent to non-specific DNA binding sites.     

Adenosine Triphosphate Stimulates Aquifex aeolicus MutL Endonuclease Activity

Background

Human PMS2 (hPMS2) homologues act to nick 5′ and 3′ to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn⁺⁺ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)₂E(X)₄E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.

Methodologies/Principal Findings

We examined the effect ATP had on the Mn⁺⁺ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL) proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6±0.08×10⁻⁵ s⁻¹ and 4.2±0.3×10⁻⁵ s⁻¹ in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X)₂E(X)₄E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.

Conclusions

ATP stimulated the Mn⁺⁺ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X)₂E(X)₄E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn⁺⁺ induced nicking activity.     

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.     

Utilization of DNA as a Sole Source of Phosphorus, Carbon, and Energy by Shewanella spp.: Ecological and Physiological Implications for Dissimilatory Metal Reduction

The solubility of orthophosphate (PO₄³⁻) in iron-rich sediments can be exceedingly low, limiting the bioavailability of this essential nutrient to microbial populations that catalyze critical biogeochemical reactions. Here we demonstrate that dissolved extracellular DNA can serve as a sole source of phosphorus, as well as carbon and energy, for metal-reducing bacteria of the genus Shewanella. Shewanella oneidensis MR-1, Shewanella putrefaciens CN32, and Shewanella sp. strain W3-18-1 all grew with DNA but displayed different growth rates. W3-18-1 exhibited the highest growth rate with DNA. While strain W3-18-1 displayed Ca²⁺-independent DNA utilization, both CN32 and MR-1 required millimolar concentrations of Ca²⁺ for growth with DNA. For S. oneidensis MR-1, the utilization of DNA as a sole source of phosphorus is linked to the activities of extracellular phosphatase(s) and a Ca²⁺-dependent nuclease(s), which are regulated by phosphorus availability. Mass spectrometry analysis of the extracellular proteome of MR-1 identified one putative endonuclease (SO1844), a predicted UshA (bifunctional UDP-sugar hydrolase/5′ nucleotidase), a predicted PhoX (calcium-activated alkaline phosphatase), and a predicted CpdB (bifunctional 2′,3′ cyclic nucleotide 2′ phosphodiesterase/3′ nucleotidase), all of which could play important roles in the extracellular degradation of DNA under phosphorus-limiting conditions. Overall, the results of this study suggest that the ability to use exogenous DNA as the sole source of phosphorus is widespread among the shewanellae, and perhaps among all prokaryotes, and may be especially important for nutrient cycling in metal-reducing environments.     

Structural Insights of the ssDNA Binding Site in the Multifunctional Endonuclease AtBFN2 from Arabidopsis thaliana

The multi S1/P1 nuclease AtBFN2 (EC 3.1.30.1) encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 3′-OH to yield 5′-nucleotides. In addition, AtBFN2′s enzymatic activity is strongly glycan dependent. Plant Zn²⁺-dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC)/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO₄ models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A₅T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn²⁺-dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex.     

Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D

The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3′-5′ proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn²⁺-dependent 3′-5′ exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.     

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.     

Structure–activity relationships in Kluyveromyces lactis γ-toxin,a eukaryal tRNA anticodon nuclease

tRNA anticodon damage inflicted by secreted ribotoxins such as Kluyveromyces lactis γ-toxin and bacterial colicins underlies a rudimentary innate immune system that distinguishes self from nonself species. The intracellular expression of γ-toxin (a 232-amino acid polypeptide) arrests the growth of Saccharomyces cerevisiae by incising a single RNA phosphodiester 3′ of the modified wobble base of tRNA^Glu. Fungal γ-toxin bears no primary structure similarity to any known nuclease and has no plausible homologs in the protein database. To gain insight to γ-toxin''s mechanism, we tested the effects of alanine mutations at 62 basic, acidic, and polar amino acids on ribotoxin activity in vivo. We thereby identified 22 essential residues, including 10 lysines, seven arginines, three glutamates, one cysteine, and one histidine (His209, the only histidine present in γ-toxin). Structure–activity relations were gleaned from the effects of 44 conservative substitutions. Recombinant tag-free γ-toxin, a monomeric protein, incised an oligonucleotide corresponding to the anticodon stem–loop of tRNA^Glu at a single phosphodiester 3′ of the wobble uridine. The anticodon nuclease was metal independent. RNA cleavage was abolished by ribose 2′-H and 2′-F modifications of the wobble uridine. Mutating His209 to alanine, glutamine, or asparagine abolished nuclease activity. We propose that γ-toxin catalyzes an RNase A-like transesterification reaction that relies on His209 and a second nonhistidine side chain as general acid–base catalysts.     

Ligation reaction specificities of an NAD(+)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus

An NAD⁺-dependent DNA ligase from the hyperthermophilic bacterium Aquifex aeolicus was cloned, expressed in Escherichia coli and purified to homogeneity. The enzyme is most active in slightly alkaline pH conditions with either Mg²⁺ or Mn²⁺ as the metal cofactor. Ca²⁺ and Ni²⁺ mainly support formation of DNA–adenylate intermediates. The catalytic cycle is characterized by a low k_cat value of 2 min^–1 with concomitant accumulation of the DNA–adenylate intermediate when Mg²⁺ is used as the metal cofactor. The ligation rates of matched substrates vary by up to 4-fold, but exhibit a general trend of T/A ≤ G/C < C/G < A/T on both the 3′- and 5′-side of the nick. Consistent with previous studies on Thermus ligases, this Aquifex ligase exhibits greater discrimination against a mismatched base pair on the 3′-side of the nick junction. The requirement of 3′ complementarity for a ligation reaction is reaffirmed by results from 1 nt insertions on either the 3′- or 5′-side of the nick. Furthermore, most of the unligatable 3′ mismatched base pairs prohibit formation of the DNA–adenylate intermediate, indicating that the substrate adenylation step is also a control point for ligation fidelity. Unlike previously studied ATP ligases, gapped substrates cannot be ligated and intermediate accumulation is minimal, suggesting that complete elimination of base pair complementarity on one side of the nick affects substrate adenylation on the 5′-side of the nick junction. Relationships among metal cofactors, ligation products and intermediate, and ligation fidelity are discussed.     

Structural and Mechanistic Insight into DNA Unwinding by Deinococcus radiodurans UvrD

DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement around duplexed DNA. Our structural data also improve our understanding of the molecular mechanisms that regulate DNA unwinding by Superfamily 1A (SF1A) helicases. Our biochemical data reveal that drUvrD is a DNA-stimulated ATPase, can translocate along ssDNA in the 3′-5′ direction and shows ATP-dependent 3′-5′, and surprisingly also, 5′-3′ helicase activity. Interestingly, we find that these translocase and helicase activities of drUvrD are modulated by the ssDNA binding protein. Analysis of drUvrD mutants indicate that the conserved β-hairpin structure of drUvrD that functions as a separation pin is critical for both drUvrD’s 3′-5′ and 5′-3′ helicase activities, whereas the GIG motif of drUvrD involved in binding to the DNA duplex is essential for the 5′-3′ helicase activity only. These special features of drUvrD may reflect its involvement in a wide range of DNA repair processes in vivo.     

The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis

The HNH motif was originally identified in the subfamily of HNH homing endonucleases, which initiate the process of the insertion of mobile genetic elements into specific sites. Several bacteria toxins, including colicin E7 (ColE7), also contain the 30 amino acid HNH motif in their nuclease domains. In this work, we found that the nuclease domain of ColE7 (nuclease-ColE7) purified from Escherichia coli contains a one-to-one stoichiometry of zinc ion and that this zinc-containing enzyme hydrolyzes DNA without externally added divalent metal ions. The apo-enzyme, in which the indigenous zinc ion was removed from nuclease-ColE7, had no DNase activity. Several divalent metal ions, including Ni²⁺, Mg²⁺, Co²⁺, Mn²⁺, Ca²⁺, Sr²⁺, Cu²⁺ and Zn²⁺, re-activated the DNase activity of the apo-enzyme to various degrees, however higher concentrations of zinc ion inhibited this DNase activity. Two charged residues located at positions close to the zinc-binding site were mutated to alanine. The single-site mutants, R538A and E542A, showed reduced DNase activity, whereas the double-point mutant, R538A + E542A, had no observable DNase activity. A gel retardation assay further demonstrated that the nuclease-ColE7 hydrolyzed DNA in the presence of zinc ions, but only bound to DNA in the absence of zinc ions. These results demonstrate that the zinc ion in the HNH motif of nuclease-ColE7 is not required for DNA binding, but is essential for DNA hydrolysis, suggesting that the zinc ion not only stabilizes the folding of the enzyme, but is also likely to be involved in DNA hydrolysis.     

Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5′-phosphate and 3′-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3′-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3′p substrate to generate an RNA 2′,3′-cyclic phosphate or convert DNA3′p to ssDNA^3′pp^5′A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3′p. These modifications of RNA and DNA 3′-phosphates are similar to the activities of RtcA, an RNA 3′-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3′p or DNA 3′p to generate the adenylated intermediate. For RNA ^3′pp^5′A, the third step involves attack of the adjacent 2′ hydroxyl to generate the RNA 2′,3′-cyclic phosphate. These steps are analogous to those in classical 5′ phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3′p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3′-phosphorylated nicks in double-stranded DNA to produce a 3′-adenylated product. These 3′-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5′Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3′-terminal phosphates.