RecG helicase promotes DNA double-strand break repair

Double-strand breaks pose a major threat to the genome and must be repaired accurately if structural and functional integrity are to be preserved. This is usually achieved via homologous recombination, which enables the ends of a broken DNA molecule to engage an intact duplex and prime synthesis of the DNA needed for repair. In Escherichia coli, repair relies on the RecBCD and RecA proteins, the combined ability of which to initiate recombination and form joint-molecule intermediates is well understood. To shed light on subsequent events, we exploited the I-SceI homing endonuclease of yeast to make breaks at I-SceI cleavage sites engineered into the chromosome. We show that survival depends on RecA and RecBCD, and that subsequent events can proceed via either of two pathways, one dependent on the RuvABC Holliday junction resolvase and the other on RecG helicase. Both pathways rely on PriA, presumably to facilitate DNA replication. We discuss the possibility that classical Holliday junctions may not be essential intermediates in repair and consider alternative pathways for RecG-dependent separation of joint molecules formed by RecA.     

ATPase cycle and DNA unwinding kinetics of RecG helicase

The superfamily 2 bacterial helicase, RecG, is a monomeric enzyme with a role in DNA repair by reversing stalled replication forks. The helicase must act specifically and rapidly to prevent replication fork collapse. We have shown that RecG binds tightly and rapidly to four-strand oligonucleotide junctions, which mimic a stalled replication fork. The helicase unwinds such DNA junctions with a step-size of approximately four bases per ATP hydrolyzed. To gain an insight into this mechanism, we used fluorescent stopped-flow and quenched-flow to measure individual steps within the ATPase cycle of RecG, when bound to a DNA junction. The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle. Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism. The data show that the chemical step of hydrolysis is the rate limiting step in the cycle and that this step is greatly accelerated by bound DNA. The ADP release rate is similar to the cleavage rate, so that bound ATP and ADP would be the main states during the ATP cycle. Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states.     

The catalytically active form of histidinol dehydrogenase from Salmonella typhimurium.

The active-enzyme-sedimentation procedure was used to identify the catalytically competent form of histidinol dehydrogenase (EC 1.1.1.23) isolated from Salmonella typhimurium. At pH 9.4 the active species has a sedimentation coefficient S20,W of 5.4S, indicating that the dimer with a mol.wt. of approx. 83 000 is the enzymically active form.     

Mechanism of translocation and kinetics of DNA unwinding by the helicase RecG

RecG is a DNA helicase involved in the repair of damage at a replication fork and catalyzes the reversal of the fork to a point beyond the damage in the template strand. It unwinds duplex DNA in reactions that are coupled to ATP hydrolysis. The kinetic mechanism of duplex DNA unwinding by RecG was analyzed using a quantitative fluorescence assay based on the process of contact quenching between Cy3 and Dabcyl groups attached to synthetic three-way DNA junctions. The data show that the protein moves at a rate of 26 bp s(-1) along the duplex DNA during the unwinding process. RecG ATPase activity during translocation indicates a constant rate of 7.6 s(-1), measured using a fluorescent phosphate sensor, MDCC-PBP. These two rates imply a movement of approximately 3 bp per ATP hydrolyzed. We demonstrate in several trapping experiments that RecG remains attached to DNA after translocation to the end of the arm of the synthetic DNA junction. ATPase activity continues after translocation is complete. Dissociation of RecG from the product DNA occurs only very slowly, suggesting strong interactions between them. The data support the idea that interactions of the duplex template arm with the protein are the major sites of binding and production of translocation.     

RecG helicase activity at three- and four-strand DNA structures.

The RecG helicase of Escherichia coli is necessary for efficient recombination and repair of DNA in vivo and has been shown to catalyse the unwinding of DNA junctions in vitro. Despite these findings, the precise role of RecG remains elusive. However, models have been proposed in which RecG promotes the resolution of linked duplexes by targeting three-strand junctions present at D-loops. One such model postulates that RecG catalyses the formation of four-strand (Holliday) junctions from three-strand junctions. To test this model, the DNA binding and unwinding activities of RecG were analysed using synthetic three- and four-strand junctions. The substrate specificity of RecG was found to depend critically on the concentrations of ATP and MgCl(2)and under certain conditions RecG preferentially unwound three-strand junction DNA. This was at least partly due to the larger inhibitory effect of MgCl(2)on the binding of four-strand as opposed to three-strand junctions by RecG. Thus RecG may be targeted to three-strand junctions in vivo whilst still being able to branch migrate the four-strand junctions formed as a result of the initial helicase reaction. The increase in the dissociation constant of RecG on conversion of a three-strand into a four-strand junction may also facilitate resolution of the four-strand junction by the RuvABC complex.     

A catalytically active high-Mr form of human cathepsin B from sputum.

A cysteine proteinase from purulent sputum was partially purified by a method involving affinity chromatography on Sepharose-aminohexanoylphenylalanylglycinaldehyde semicarbazone. It was immunologically related to lysosomal cathepsin B from human liver and was similar in many, but not all, other aspects. It was catalytically active, as demonstrated by active-site-directed radioiodination, and hydrolysed three cathepsin B substrates, two with Km values similar to those of lysosomal cathepsin B. In addition, the rates of inactivation of the sputum and lysosomal forms of the enzyme by L-3-carboxy-2,3-transepoxypropionyl-leucylamido(4-guanidino) butane (Compound E-64) were very similar. However, the sputum enzyme differed from lysosomal cathepsin B in the following respects. Inhibition by chicken cystatin was much weaker for sputum cathepsin B than for the lysosomal enzyme. Sputum cathepsin B had greater stability at pH 7.5 and a higher apparent Mr, even after deglycosylation, than lysosomal cathepsin B. We conclude that the form of cathepsin B found in sputum is probably a truncated form of human procathepsin B, with some differences in properties that could be of physiological importance.     

DNA binding and helicase domains of the Escherichia coli recombination protein RecG.

The Escherichia coli RecG protein is a unique junction-specific helicase involved in DNA repair and recombination. The C-terminus of RecG contains motifs conserved throughout a wide range of DNA and RNA helicases and it is thought that this C-terminal half of RecG contains the helicase active site. However, the regions of RecG which confer junction DNA specificity are unknown. To begin to assign structure-function relationships within RecG, a series of N- and C-terminal deletions have been engineered into the protein, together with an N-terminal histidine tag fusion peptide for purification purposes. Junction DNA binding, unwinding and ATP hydrolysis were disrupted by mutagenesis of the N-terminus. In contrast, C-terminal deletions moderately reduced junction DNA binding but almost abolished unwinding. These data suggest that the C-terminus does contain the helicase active site whereas the N-terminus confers junction DNA specificity.     

Crystallization of a soluble,catalytically active form of Escherichia coli leader peptidase

Leader peptidase, a novel serine protease in Escherichia coli, catalyzes the cleavage of the amino-terminal leader sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Here, we report a procedure for the purification and the crystallization of a soluble non-membrane-bound form of leader peptidase (Δ2-75). Crystals were obtained by the sitting-drop vapor diffusion technique using ammonium dihydrogen phosphate as the precipitant. Interestingly, we have found that the presence of the detergent Triton X-100 is required to obtain crystals sufficiently large for X-ray analysis. The crystals belong to the tetragonal space group P4₂2₁2, with unit cell dimensions of a = b = 115 Å and c = 100 Å, and contain 2 molecules per asymmetric unit. This is the first report of the crystallization of a leader (or signal) peptidase. © 1995 Wiley-Liss, Inc.     

IS911 partial transposition products and their processing by the Escherichia coli RecG helicase

Insertion of bacterial insertion sequence IS911 can often be directed to sequences resembling its ends. We have investigated this type of transposition and shown that it can occur via cleavage of a single end and its targeted transfer next to another end. The single end transfer (SET) events generate branched DNA molecules that contain a nicked Holliday junction and can be considered as partial transposition products. Our results indicate that these can be processed by the Escherichia coli host independently of IS911-encoded proteins. Such resolution depends on the presence of homologous DNA regions neighbouring the cross-over point in the SET molecule. Processing is often accompanied by sequence conversion between donor and target sequences, suggesting that branch migration is involved. We show that resolution is greatly reduced in a recG host. Thus, the branched DNA-specific helicase, RecG, involved in processing of potentially lethal DNA structures such as stalled replication forks, also intervenes in the resolution of partial IS911 transposition products.     

Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: a comparison with catalytically inactive form

We report the accumulation of an acid unfolded (UA) state and a molten globule (MG) state in the acid induced unfolding pathway of unmodified preparation of stem bromelain (SB) [EC 3.4.22.32], a cystein protease from Ananas cosmosus. The conformation of SB was examined over the pH 0.8-3 regions by circular dichroism, tryptophanyl fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, and tryptophanyl fluorescence quenching study. The pH 0.8-3.0 regions were selected to study the acid induced unfolding of SB because no autolysis of the enzyme was observed in these pH regions. The results show that SB at pH 2.0 is maximally unfolded and characterizes by significant loss of secondary structure ( approximately 80%) and almost complete loss of tertiary contacts. However, on further decreasing the pH to 0.8 a MG state was observed, with secondary structure content similar to that of native protein but no tertiary structure. We also made a comparative study of these acid induced states of SB with acid induced states of modified stem bromelain (mSB), reported by our group earlier [Eur. J. Biochem. (2002) 269, 47-52]. We have shown that modification of SB for inactivation significantly affects the N-UA transition but neither affects the UA-MG transition nor the stability of the MG state.     

Characterisation of Bacillus stearothermophilus PcrA helicase: evidence against an active rolling mechanism.

PcrA from Bacillus stearothermophilus is a DNA helicase for which, despite the availability of a crystal structure, there is very little biochemical information. We show that the enzyme has a broad nucleotide specificity, even being able to hydrolyse ethenonucleotides, and is able to couple the hydrolysis to unwinding of DNA substrates. In common with the Escherichia coli helicases Rep and UvrD, PcrA is a 3'-5' helicase but at high protein concentrations it can also displace a substrate with a 5' tail. However, in contrast to Rep and UvrD, we do not see any evidence for dimerisation of the protein even in the presence of DNA. The enzyme shows a specificity for the DNA substrate in gel mobility assays, with the preferred substrate being one with both single and double stranded regions of DNA. We propose that these data, together with existing structural evidence, support an inchworm rather than a rolling model for 3'-5' helicase activity.     

Interplay between DNA replication, recombination and repair based on the structure of RecG helicase

Recent studies in Escherichia coli indicate that the interconversion of DNA replication fork and Holliday junction structures underpins chromosome duplication and helps secure faithful transmission of the genome from one generation to the next. It facilitates interplay between DNA replication, recombination and repair, and provides means to rescue replication forks stalled by lesions in or on the template DNA. Insight into how this interconversion may be catalysed has emerged from genetic, biochemical and structural studies of RecG protein, a member of superfamily 2 of DNA and RNA helicases. We describe how a single molecule of RecG might target a branched DNA structure and translocate a single duplex arm to drive branch migration of a Holliday junction, interconvert replication fork and Holliday junction structures and displace the invading strand from a D loop formed during recombination at a DNA end. We present genetic evidence suggesting how the latter activity may provide an efficient pathway for the repair of DNA double-strand breaks that avoids crossing over, thus facilitating chromosome segregation at cell division.     

A Dimer of Escherichia coli UvrD is the active form of the helicase in vitro

The Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized in vitro UvrD-catalyzed unwinding of a series of 18 bp duplex DNA substrates with 3' single-stranded DNA (ssDNA) tails ranging in length from two to 40 nt. Single turnover DNA-unwinding experiments were performed using chemical quenched flow methods, as a function of both [UvrD] and [DNA] under conditions such that UvrD-DNA binding is stoichiometric. Although a single UvrD monomer binds tightly to the single-stranded/double-stranded DNA (dsDNA) junction if the 3' ssDNA tail is at least four nt, no unwinding was observed for DNA substrates with tail-lengths /=12 nt, and the unwinding amplitude displays a sigmoidal dependence on [UvrD(tot)]/[DNA(tot)]. Quantitative analysis of these data indicates that a single UvrD monomer bound at the ssDNA/dsDNA junction of any DNA substrate, independent of 3' ssDNA tail length, is not competent to fully unwind even a short 18 bp duplex DNA, and that two UvrD monomers must bind the DNA substrate in order to form a complex that is able to unwind short DNA substrates in vitro. Other proteins, including a mutant UvrD with no ATPase activity as well as a monomer of the structurally homologous E.coli Rep helicase, cannot substitute for the second UvrD monomer, suggesting a specific interaction between two UvrD monomers and that both must be able to hydrolyze ATP. Initiation of DNA unwinding in vitro appears to require a dimeric UvrD complex in which one subunit is bound to the ssDNA/dsDNA junction, while the second subunit is bound to the 3' ssDNA tail.     

Sedimentation study of a catalytically active form of rabbit muscle phosphofructokinase at pH 8.55

The enzymatic active form of rabbit muscle phosphofructokinase (PFK) was observed directly by using the method of reacting or active enzyme centrifugation (AEC). These studies were performed in two assay systems: a coupled enzyme and a pH-dependent dye-linked system in glycylglycine buffer at pH 8.55 and 23 +/- 1 degree C. The sedimenting band of PFK was stabilized by three solvent systems: 50% (v/v) D2O, 10% (w/v) sucrose, and 4% (v/v) or 10% (v/v) glycerol. The active PFK species sediments as a single component with a sedimentation coefficient of 12.4 +/- 0.5 S, after correcting for protein--solvent interactions. Although PFK may undergo association--dissociation, there is no observable change in the value of s20,w over a 57-fold range of protein concentration. Throughout this range only a single active species of PFK was observed, and within an experimental uncertainty of +/- 10%, the enzymatic activity observed in the sedimentation studies accounts for the total enzymatic activity observed in the steady-state kinetics. Partially purified PFK was subjected to AEC analysis. Results reveal the presence of again a single active form sedimenting at the same rate as the purified enzyme. Results from sedimentation velocity studies indicate that the stabilizing solvents employed in AEC enhance the self-association of PFK. However, such an enhancement alone cannot account for the observation of a single active species with a sedimentation coefficient of 12.4 S. The interactions between solvent additives and PFK were studied by density measurements and by the application of multicomponent theory. Results from such a preferential solvent interaction study indicate that PFK is preferentially hydrated in the presence of sucrose or glycerol. The enhancement of PFK self-association is most likely due to a nonspecific solvent--protein interaction.     

Synthesis of catalytically active form III ribulose 1,5-bisphosphate carboxylase/oxygenase in archaea

Ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the biological reduction and assimilation of carbon dioxide gas to organic carbon; it is the key enzyme responsible for the bulk of organic matter found on earth. Until recently it was believed that there are only two forms of RubisCO, form I and form II. However, the recent completion of several genome-sequencing projects uncovered open reading frames resembling RubisCO in the third domain of life, the archaea. Previous work and homology comparisons suggest that these enzymes represent a third form of RubisCO, form III. While earlier work indicated that two structurally distinct recombinant archaeal RubisCO proteins catalyzed bona fide RubisCO reactions, it was not established that the rbcL genes of anaerobic archaea can be transcribed and translated to an active enzyme in the native organisms. In this report, it is shown not only that Methanococcus jannaschii, Archaeoglobus fulgidus, Methanosarcina acetivorans, and Methanosarcina barkeri possess open reading frames with the residues required for catalysis but also that the RubisCO protein from these archaea accumulates in an active form under normal growth conditions. In addition, the form III RubisCO gene (rbcL) from M. acetivorans was shown to complement RubisCO deletion strains of Rhodobacter capsulatus and Rhodobacter sphaeroides under both photoheterotrophic and photoautotrophic growth conditions. These studies thus indicate for the first time that archaeal form III RubisCO functions in a physiologically significant fashion to fix CO(2). Furthermore, recombinant M. jannaschii, M. acetivorans, and A. fulgidus RubisCO possess unique properties with respect to quaternary structure, temperature optima, and activity in the presence of molecular oxygen compared to the previously described Thermococcus kodakaraensis and halophile proteins.     

Purification of the catalytically active phosphorylated form of insulin receptor kinase by affinity chromatography with O-phosphotyrosyl-binding antibodies

The catalytically active, tyrosyl-phosphorylated form of insulin receptor kinase was isolated from human placenta by a procedure which exploits the propensity for the intact alpha 2 beta 2 form of insulin receptor to undergo insulin-promoted autophosphorylation at tyrosyl residues and concomitant activation as a tyrosyl kinase. Purification of tyrosyl-phosphorylated insulin receptor was effected by adsorption on and elution (with a hapten) from a column of O-phosphotyrosyl-binding antibody immobilized on protein A-Sepharose (Ab-protein A). The starting material for the purification process was protein which had been solubilized from placental membranes and purified by chromatography on immobilized wheat germ agglutinin. After chromatography on Ab-protein A to remove preexisting O-phosphotyrosyl-containing proteins, the fraction which did not adsorb to the Ab-protein A column was incubated with insulin and briefly treated with ATP so as to maximize selective autophosphorylation of insulin receptor. This material was then subjected to chromatography on Ab-protein A. Although the amount of the intact alpha 2 beta 2 form of insulin receptor present in the starting material was only a small fraction of the protein (approximately 0.2%) and only approximately 20% of the insulin-binding forms of the receptor present, it was eluted (with 10 mM p-nitrophenyl phosphate) from the column in greater than or equal to 80% purity. Chromatography on Ab-protein A appears to have an advantage over the alternative affinity chromatographic procedures which utilize immobilized insulin or antiinsulin receptor antibody to adsorb insulin receptor, since these procedures do not resolve the intact alpha 2 beta 2 form of insulin receptor from the nicked insulin-binding forms of the receptor which do not undergo insulin promoted autophosphorylation.     

Interplay between Bacillus subtilis RecD2 and the RecG or RuvAB helicase in recombinational repair

Bacillus subtilis AddAB, RecS, RecQ, PcrA, HelD, DinG, RecG, RuvAB, PriA and RecD2 are genuine recombinational repair enzymes, but the biological role of RecD2 is poorly defined. A ΔrecD2 mutation sensitizes cells to DNA-damaging agents that stall or collapse replication forks. We found that this ΔrecD2 mutation impaired growth, and that a mutation in the pcrA gene (pcrA596) relieved this phenotype. The ΔrecD2 mutation was not epistatic to ΔaddAB, ΔrecQ, ΔrecS, ΔhelD, pcrA596 and ΔdinG, but epistatic to recA. Specific RecD2 degradation caused unviability in the absence of RecG or RuvAB, but not on cells lacking RecU. These findings show that there is notable interplay between RecD2 and RecG or RuvAB at arrested replication forks, rather than involvement in processing Holliday junctions during canonical double strand break repair. We propose that there is a trade-off for efficient genome duplication, and that recombinational DNA helicases directly or indirectly provide the cell with the means to tolerate chromosome segregation failures.     

Immobilization of catalytically active thromboxane synthase

Thromboxane synthase has been immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme is catalytically active and has a slightly lower apparent Km for PGH2 than the detergent-solubilized enzyme. However, both imidazole- and pyridine-based inhibitors are equally effective in inhibiting the immobilized and solubilized enzyme preparations. Although the immobilized enzyme appears to be less stable than the solubilized enzyme it is sufficiently stable to be used as a model for studying the properties of the enzyme.