Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family

Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5′-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3nt from the 3′-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.     

Uni-site catalysis by Escherichia coli F1-ATPase with different numbers of bound nucleotides

We prepared two types of E. coli F1 by slightly different gel filtration procedures of the purified F1: F1(II) contained about 2 mol, and F1(V) about 5 mol of bound adenine nucleotides per mol of the enzyme. Thus F1(II) had more than 2, possibly 3, vacant catalytic sites, while F1(V) had less than one vacant catalytic site. The rate of ATP hydrolysis in uni-site catalysis (in the presence of inorganic phosphate) was about 3-fold higher with F1(II) than with F1(V), suggesting that ADP and inorganic phosphate bound at the catalytic sites of F1(V) changed the kinetics of uni-site catalysis significantly.     

Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication

DNA replication and cellular survival requires efficient removal of RNA primers during lagging strand DNA synthesis. In eukaryotes, RNA primer removal is initiated by type 2 RNase H, which specifically cleaves the RNA portion of an RNA-DNA/DNA hybrid duplex. This conserved type 2 RNase H family of replicative enzymes shares little sequence similarity with the well-characterized prokaryotic type 1 RNase H enzymes, yet both possess similar enzymatic properties. Crystal structures and structure-based mutational analysis of RNase HII from Archaeoglobus fulgidus, both with and without a bound metal ion, identify the active site for type 2 RNase H enzymes that provides the general nuclease activity necessary for catalysis. The two-domain architecture of type 2 RNase H creates a positively charged binding groove and links the unique C-terminal helix-loop-helix cap domain to the active site catalytic domain. This architectural arrangement apparently couples directional A-form duplex binding, by a hydrogen-bonding Arg-Lys phosphate ruler motif, to substrate-discrimination, by a tyrosine finger motif, thereby providing substrate-specific catalytic activity. Combined kinetic and mutational analyses of structurally implicated substrate binding residues validate this binding mode. These structural and mutational results together suggest a molecular mechanism for type 2 RNase H enzymes for the specific recognition and cleavage of RNA in the RNA-DNA junction within hybrid duplexes, which reconciles the broad substrate binding affinity with the catalytic specificity observed in biochemical assays. In combination with a recent independent structural analysis, these results furthermore identify testable molecular hypotheses for the activity and function of the type 2 RNase H family of enzymes, including structural complementarity, substrate-mediated conformational changes and coordination with subsequent FEN-1 activity.     

Catalytic site occupancy during ATP hydrolysis by MF1-ATPase. Evidence for alternating high affinity sites during steady-state turnover

The mechanism of ATP hydrolysis by the solubilized mitochondrial ATPase (MF1) has been studied under conditions where catalytic turnover occurs at one site, uni-site catalysis (obtained when enzyme is in excess of substrate), or at two sites, bi-site catalysis (obtained when substrate is in excess of enzyme). Pulse-chase experiments support the conclusion that the sites which participate in bi-site catalysis are the same as those which participate in uni-site catalysis. Upon addition of ATP in molar excess to MF1, label that was bound under uni-site conditions dissociates at a rate equal to the rate of bi-site catalysis. Similarly, when medium ATP is removed, label that was bound under bi-site conditions dissociates at a rate equal to the rate of uni-site catalysis. Evidence that a high affinity catalytic site equivalent to the one observed under uni-site conditions participates as an intermediate in bi-site catalysis includes the demonstration of full occupancy of a catalytically competent site during steady-state turnover at nanomolar concentrations of ATP. Improved measurements of the interaction of ADP at a high affinity catalytic site have lead to the revision of several of the rate constants that define uni-site catalysis. The rate constant for unpromoted dissociation of ADP is equal to that for Pi (4 X 10(-3) s-1). The rate of binding ADP at a high affinity chaseable site (Kd = 1 nM) is equal to the rate of binding ATP (4 X 10(6) M-1 s-1). The rate of catalysis obtained when substrate binding at one site promotes product release from an adjacent site (bi-site catalysis) is up to 100,000-fold faster than unpromoted product release (uni-site catalysis).     

The activity of the EcoRV restriction endonuclease is influenced by flanking DNA sequences both inside and outside the DNA-protein complex.

The EcoRV restriction endonuclease cleaves DNA not only at its recognition sequence but also at most other sequences that differ from the recognition site by one base pair. Compared to the reaction at the recognition site, the reactions at noncognate sites are slow but 1 out of the 12 noncognate sites on the plasmid pAT153 is cleaved more than 50 times faster than any other. The increase in the reaction rate at the preferred noncognate site, relative to other sites, was caused by the DNA sequences in the 4 base pairs from either side of the site. For enhanced activity by EcoRV, particular bases were needed immediately adjacent to the site, inside the DNA-protein complex. At these loci, the protein interacts with the phosphate groups in the DNA and the flanking sequence may control the activity of the enzyme by determining the conformation of the DNA, thus aligning the phosphate contacts. But the preferential cleavage also depended on sequences further away from the site, at loci outside the complex. At external positions, beyond the reach of the protein, the EcoRV enzyme required flanking sequences that give rise to flexibility in DNA conformation. These may facilitate the distortion of the DNA required for catalysis by EcoRV.     

Enzymatic catalysis in crystals of Escherichia coli maltodextrin phosphorylase

The bacterial enzyme maltodextrin phosphorylase (MalP) catalyses the phosphorolysis of an alpha-1,4-glycosidic bond in maltodextrins, removing the non-reducing glucosyl residues of linear oligosaccharides as glucose-1-phosphate (Glc1P). In contrast to the well-studied muscle glycogen phosphorylase (GP), MalP exhibits no allosteric properties and has a higher affinity for linear oligosaccharides than GP. We have used MalP as a model system to study catalysis in the crystal in the direction of maltodextrin synthesis. The 2.0A crystal structure of the MalP/Glc1P binary complex shows that the Glc1P substrate adopts a conformation seen previously with both inactive and active forms of mammalian GP, with the phosphate group not in close contact with the 5'-phosphate group of the essential pyridoxal phosphate (PLP) cofactor. In the active MalP enzyme, the residue Arg569 stabilizes the negative-charged Glc1P, whereas in the inactive form of GP this key residue is held away from the catalytic site by loop 280s and an allosteric transition of the mammalian enzyme is required for activation. The comparison between MalP structures shows that His377, through a hydrogen bond with the 6-hydroxyl group of Glc1P substrate, triggers a conformational change of the 380s loop. This mobile region folds over the catalytic site and contributes to the specific recognition of the oligosaccharide and to the synergism between substrates in promoting the formation of the MalP ternary complex. The structures solved after the diffusion of oligosaccharides (either maltotetraose, G4 or maltopentaose, G5) into MalP/Glc1P crystals show the formation of phosphate and elongation of the oligosaccharide chain. These structures, refined at 1.8A and at 2.2A, confirm that only when an oligosaccharide is bound to the catalytic site will Glc1P bend its phosphate group down so it can contact the PLP 5' phosphate group and promote catalysis. The relatively large oligosaccharide substrates can diffuse quickly into the MalP/Glc1P crystals and the enzymatic reaction can occur without significant crystal damage. These structures obtained before and after catalysis have been used as frames of a molecular movie. This movie reveals the relative positions of substrates in the catalytic channel and shows a minimal movement of the protein, involving mainly Arg569, which tracks the substrate phosphate group.     

The defective proton-ATPase of uncD mutants of Escherichia coli. Two mutations which affect the catalytic mechanism

The catalytic characteristics of F1-ATPases from uncD412 and uncD484 mutant strains of Escherichia coli were studied in order to understand how these beta-subunit mutations cause defective catalysis. Both mutant enzymes showed reduced affinity for ATP at the first catalytic site. While uncD412 F1 was similar to normal in other aspects of single site catalysis, uncD484 F1 showed a Keq of bound reactants greatly biased toward bound substrate ATP and an abnormally fast rate of Pi release. Impairment of productive catalytic cooperativity was the major cause of the reduced steady state ("multisite") catalytic rate in both mutant enzymes. Addition of excess ATP to saturate second and/or third catalytic sites did promote ATP hydrolysis and product release at the first catalytic site of uncD412 F1, but the multisite turnover rate was significantly slower than normal. In contrast, with uncD484 F1, addition of excess ATP induced rapid release of ATP from the first catalytic site and so productive catalytic cooperativity was almost completely absent. The results show that both mutations affect properties of the catalytic site and catalytic site cooperativity and further that the relatively more severe uncD484 mutation affects a residue which acts as a determinant of the fate of bound substrate ATP during promotion of catalysis. Taken together with previous studies of uncA mutant F1-ATPases (Wise, J. G., Latchney, L. R., Ferguson, A. M., and Senior, A. E. (1984) Biochemistry 23, 1426-1432) the results indicate that catalytic site cooperativity in F1-ATPases involves concerted beta-alpha-beta intersubunit communication between catalytic sites on the beta-subunits.     

Escherichia coli ATP synthase alpha subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state

The three catalytic sites of the F(O)F(1) ATP synthase interact through a cooperative mechanism that is required for the promotion of catalysis. Replacement of the conserved alpha subunit Arg-376 in the Escherichia coli F(1) catalytic site with Ala or Lys resulted in turnover rates of ATP hydrolysis that were 2 x 10(3)-fold lower than that of the wild type. Mutant enzymes catalyzed hydrolysis at a single site with kinetics similar to that of the wild type; however, addition of excess ATP did not chase bound ATP, ADP, or Pi from the catalytic site, indicating that binding of ATP to the second and third sites failed to promote release of products from the first site. Direct monitoring of nucleotide binding in the alphaR376A and alphaR376K mutant F(1) by a tryptophan in place of betaTyr-331 (Weber et al. (1993) J. Biol. Chem. 268, 20126-20133) showed that the catalytic sites of the mutant enzymes, like the wild type, have different affinities and therefore, are structurally asymmetric. These results indicate that alphaArg-376, which is close to the beta- or gamma-phosphate group of bound ADP or ATP, respectively, does not make a significant contribution to the catalytic reaction, but coordination of the arginine to nucleotide filling the low-affinity sites is essential for promotion of rotational catalysis to steady-state turnover.     

Adenine nucleotide binding sites on beef heart F1-ATPase. Asymmetry and subunit location

Previously we have shown that beef heart mitochondrial F1 contains a total of six adenine nucleotide binding sites. Three "catalytic" sites exchange bound ligand rapidly during hydrolysis of MgATP, whereas three "noncatalytic" sites do not. The noncatalytic sites behave asymmetrically in that a single site releases bound ligand upon precipitation of F1 with ammonium sulfate. In the present study, we find this same site to be the only noncatalytic site that undergoes rapid exchange of bound ligand when F1 is incubated in the presence of EDTA at pH 8.0. Following 1000 catalytic turnovers/F1, the site retains the unique capacity for EDTA-induced exchange, indicating that the asymmetric determinants are permanent and that the three noncatalytic sites on soluble F1 do not pass through equivalent states during catalysis. Measurements of the rate of ligand binding at the unique noncatalytic site show that uncomplexed nucleotide binds preferentially. At pH 7.5, in the presence of Mg2+, the rate constant for ADP binding is 9 X 10(3) M-1 s-1 and for dissociation is 4 X 10(-4) s-1 to give a Kd = 50 nM. The rate of dissociation is 10 times faster in the presence of EDTA or during MgATP hydrolysis, and it increases rapidly at pH below 7. EDTA-induced exchange is inhibited by Mg2+, Mn2+, Co2+, and Zn2+ but not by Ca2+ and is unaffected by dicyclohexylcarbodiimide modification. The unique noncatalytic site binds 2-azido-ADP. Photolysis results in the labeling of the beta subunit. Photolabeling of a single high-affinity catalytic site under conditions for uni-site catalysis also results in the labeling of beta, but a different pattern of labeled peptides is obtained in proteolytic digests. The results demonstrate the presence of two different nucleotide binding domains on the beta subunit of mitochondrial F1.     

Mechanism of allosteric regulation of Dnmt1's processivity

We have analyzed the relationship between the allosteric regulation and processive catalysis of DNA methyltransferase 1 (Dnmt1). Processivity is described quantitatively in terms of turnover rate, DNA dissociation rate, and processivity probability. Our results provide further evidence that the active site and the allosteric sites on Dnmt1 can bind DNA independently. Dnmt1's processive catalysis on unmethylated DNA is partially inhibited when the allosteric site binds unmethylated DNA and fully inhibited when the allosteric site binds a single-stranded oligonucleotide inhibitor. The partial inhibition by unmethylated DNA is caused by a decrease in the turnover rate and an increase in the substrate DNA dissociation rate. Processive catalysis with premethylated DNA is not affected if the allosteric site is exposed to premethylated DNA but is fully inhibited if the allosteric site binds unmethylated DNA or poly(dA-dT). In sum, the occupancy of the allosteric site modulates the enzyme's commitment to catalysis, which reflects the nature of the substrate and the DNA bound at the allosteric site. Our in vitro results are consistent with the possibility that the processive action of Dnmt1 may be regulated in vivo by specific regulatory nucleic acids such as DNA, RNA, or poly(ADP-ribose).     

Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase, Ser148----Ala, was produced by oligonucleotide-directed mutagenesis. The study of the catalytic properties of this mutant has shown that this mutation significantly affects the Michaelis constant of inorganic phosphate and to a lesser extent that of 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate. This result is consistent with model-building studies which show that, for the phosphorylation step of catalysis, inorganic phosphate must bind to the anion recognition site designated Pi with the C(3) phosphate of the acyl-enzyme intermediate in the alternative anion site Ps. Studies of the enantiomeric specificity using D- and L-glyceraldehyde as substrates show that the hydroxyl group of Ser148, combined with the presence of the C(3) phosphate of the substrate, enhances stereospecificity as well as catalysis. However, the stereospecific effect cannot be a consequence of the direct interaction of Ser148 with the C(2)-hydroxyl of the substrate. The changed Km for glyceraldehyde-3-phosphate suggests that the initial step of hemithioacetal formation may take place with its C(3) phosphate bound in the Pi site. This supports the molecular mechanism proposed by Moody (1984). Therefore, catalysis could be enhanced through interactions of the serine hydroxyl group not only with inorganic phosphate but also with the C(3) phosphate of glyceraldehyde-3-phosphate.     

The mechanism of action of mitochondrial ATPase (ATP synthase)

The F₁ part of the ATP synthase contains 6 nucleotide binding sites, four of which can be occupied and covalently labeled with 8-azido-adenine nucleotides. The other two sites contain tightly bound nucleotides that cannot be replaced by 8-azido-adenine nucleotides. Of the four exchangeable sites two are directly ivolved in catalysis and these are located on -subunits, while the other two are located at - interfaces and have probably a regulatory role by influencing the affinity of the catalytic sites for substrate and product. When only one catalytic site contains substrate the affinity is very high, the rate of hydrolysis is slow, and the dissociation of products is even slower (single-site catalysis). When the second site also becomes occupied, the affinity decreases enormously, and the rate of hydrolysis and dissociation of products increases several orders of magnitude. When, however, the second site is occupied by substrate in such a way that turnover is not possible at this site (e.g., covalent linkage of nitreno-ATP), the first site is no longer active, apart from the very slow single-site catalysis. The two nonexchangeable, tightly bound nucleotides that cannot be replaced by 8-azido-nucleotides, can be replaced by 2-azido-nucleotides, due to their anticonfiguration. This anticonfiguration of the substrate is also required for binding with high affinity to a catalytic site. A picture emerges in which one of the three - pairs of F₁ contains tightly bound, nonexchangeable nucleotides, while the other two contain both one catalytic site (on ) and one regulatory site (at the - interface). Cooperativity exists both between the two catalytic sites and between the catalytic and the regulatory sites.     

Structural characterization of human 8-oxoguanine DNA glycosylase variants bearing active site mutations

The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5' to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is "hardwired." Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F(*149)) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F(*292)) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.     

Tryptophan-free human PNP reveals catalytic site interactions

Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Trp residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The (13)C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steady-state kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate-phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Trp residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes.     

Catalytic site forms and controls in ATP synthase catalysis

A suggested minimal scheme for substrate binding by and interconversion of three forms of the catalytic sites of the ATP synthase is presented. Each binding change, that drives simultaneous interchange of the three catalytic site forms, requires a 120 degrees rotation of the gamma with respect to the beta subunits. The binding of substrate(s) at two catalytic sites is regarded as sufficing for near maximal catalytic rates to be attained. Although three sites do not need to be filled for rapid catalysis, during rapid bisite catalysis some enzyme may be transiently present with three sites filled. Forms with preferential binding for ADP and P(i) or for ATP are considered to arise from the transition state and participate in other steps of the catalysis. Intermediate forms and steps that may be involved are evaluated. Experimental evidence for energy-dependent steps and for control of coupling to proton translocation and transition state forms are reviewed. Impact of relevant past data on present understanding of catalytic events is considered. In synthesis a key step is suggested in which proton translocation begins to deform an open site so as to increase the affinity for ADP and P(i), that then bind and pass through the transition state, and yield tightly bound ATP in one binding change. ADP binding appears to be a key parameter controlling rotation during synthesis. In hydrolysis ATP binding to a loose site likely precedes any proton translocation, with proton movement occurring as the tight site form develops. Aspects needing further study are noted. Characteristics of the related MgADP inhibition of the F(1) ATPases that have undermined many observations are summarized, and relations of three-site filling to catalysis are assessed.     

Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease

Mutational analysis of amino acids at the periphery of the EcoRV endonuclease active site suggests that moderate-range electrostatic effects play a significant role in modulating the efficiency of phosphoryl transfer. Asp36 and Lys38 located on minor-groove binding surface loops approach within 7-9 A of the scissile phosphates of the DNA. While the rates of single-site mutations removing the carboxylate or amine moieties at these positions are decreased 10(3)-10(5)-fold compared to that of wild-type EcoRV, we find that double mutants which rebalance the charge improve catalysis by up to 500-fold. Mutational analysis also suggests that catalytic efficiency is influenced by Lys173, which is buried at the base of a deep depression penetrating from a distal surface of the enzyme. The Lys173 amine group lies just 6 A from the amine group of the conserved essential Lys92 side chain in the active site. Kinetic and crystallographic analyses of the EcoRV E45A mutant enzyme further show that the Glu45 carboxylate group facilitates an extensive set of conformational transitions which occur upon DNA binding. The crystal structure of E45A bound to DNA and Mn2+ ions reveals significant conformational alterations in a small alpha-helical portion of the dimer interface located adjacent to the DNA minor groove. This leads to a tertiary reorientation of the two monomers as well as shifting of the key major-groove binding recognition loops. Because the Glu45 side chain does not appear to play a direct structural role in maintaining the active site, these rearrangements may instead originate in an altered electrostatic potential caused by removal of the negative charge. A Mn2+ binding site on the scissile phosphate is also disrupted in the E45A structure such that inner-sphere metal interactions made by the scissile DNA phosphate and conserved Asp90 carboxylate are each replaced with water molecules in the mutant. These findings argue against a proposed role for Asp36 as the general base in EcoRV catalysis, and reveal that the induced-fit conformational changes necessary for active site assembly and metal binding are significantly modulated by the electrostatic potential in this region.     

The McrBC endonuclease translocates DNA in a reaction dependent on GTP hydrolysis.

McrBC specifically recognizes and cleaves methylated DNA in a reaction dependent on GTP hydrolysis. DNA cleavage requires at least two recognition sites that are optimally separated by 40-80 bp, but can be spaced as far as 3 kb apart. The nature of the communication between two recognition sites was analyzed on DNA substrates containing one or two recognition sites. DNA cleavage of circular DNA required only one methylated recognition site, whereas the linearized form of this substrate was not cleaved. However, the linearized substrate was cleaved if a Lac repressor was bound adjacent to the recognition site. These results suggest a model in which communication between two remote sites is accomplished by DNA translocation rather than looping. A mutant protein with defective GTPase activity cleaved substrates with closely spaced recognition sites, but not substrates where the sites were further apart. This indicates that McrBC translocates DNA in a reaction dependent on GTP hydrolysis. We suggest that DNA cleavage occurs by the encounter of two DNA-translocating McrBC complexes, or can be triggered by non-specific physical obstacles like the Lac repressor bound on the enzyme's path along DNA. Our results indicate that McrBC belongs to the general class of DNA "motor proteins", which use the free energy associated with nucleoside 5'-triphosphate hydrolysis to translocate along DNA.     

Fidelity of DNA sequence recognition by the SfiI restriction endonuclease is determined by communications between its two DNA-binding sites

The SfiI restriction endonuclease is a tetramer in which two subunits form a dimeric unit that contains one DNA binding cleft and the other two subunits contain a second cleft on the opposite side of the protein. Full activity requires both clefts to be filled with its recognition sequence: SfiI has low activity when bound to one site. The ability of SfiI to cleave non-cognate sites, one base pair different from the true site, was initially tested on substrates that lacked specific sites but which contained either one or multiple non-cognate sites. No cleavage of the DNA with one non-cognate site was detected, while a small fraction of the DNA with multiple sites was nicked. The alternative sequences were, however, cleaved in both strands, albeit at low levels, when the DNA also carried either a recognition site for SfiI or the termini generated by SfiI. Further tests employed a mutant of SfiI, altered at the dimer interface, which was known to be more active than wild-type SfiI when bound to a single site. This mutant similarly failed to cleave DNA with one non-cognate site, but cleaved the substrates with multiple non-cognate sites more readily than did the native enzyme. To cleave additional sites, SfiI thus needs to interact concurrently with either two non-cognate sites or one non-cognate and one cognate site (or the termini thereof), yet this arrangement is still restrained from cleaving the alternative site unless the communication pathway between the two DNA-binding clefts is disrupted.     

Protein assembly and DNA looping by the FokI restriction endonuclease

The FokI restriction endonuclease recognizes an asymmetric DNA sequence and cuts both strands at fixed positions upstream of the site. The sequence is contacted by a single monomer of the protein, but the monomer has only one catalytic centre and forms a dimer to cut both strands. FokI is also known to cleave DNA with two copies of its site more rapidly than DNA with one copy. To discover how FokI acts at a single site and how it acts at two sites, its reactions were examined on a series of plasmids with either one recognition site or with two sites separated by varied distances, sometimes in the presence of a DNA-binding defective mutant of FokI. These experiments showed that, to cleave DNA with one site, the monomer bound to that site associates via a weak protein–protein interaction with a second monomer that remains detached from the recognition sequence. Nevertheless, the second monomer catalyses phosphodiester bond hydrolysis at the same rate as the DNA-bound monomer. On DNA with two sites, two monomers of FokI interact strongly, as a result of being tethered to the same molecule of DNA, and sequester the intervening DNA in a loop.     

Homology modeling and molecular dynamics simulations of HgiDII methyltransferase in complex with DNA and S-adenosyl-methionine: Catalytic mechanism and interactions with DNA

M.HgiDII is a methyltransferase (MTase) from Herpetosiphon giganteus that recognizes the sequence GTCGAC. This enzyme belongs to a group of MTases that share a high degree of amino acid similarity, albeit none of them has been thoroughly characterized. To study the catalytic mechanism of M.HgiDII and its interactions with DNA, we performed molecular dynamics simulations with a homology model of M.HgiDII complexed with DNA and S-adenosyl-methionine. Our results indicate that M.HgiDII may not rely only on Glu119 to activate the cytosine ring, which is an early step in the catalysis of cytosine methylation; apparently, Arg160 and Arg162 may also participate in the activation by interacting with cytosine O2. Another residue from the catalytic site, Val118, also played a relevant role in the catalysis of M.HgiDII. Val118 interacted with the target cytosine and kept water molecules from accessing the region of the catalytic pocket where Cys79 interacts with cytosine, thus preventing water-mediated disruption of interactions in the catalytic site. Specific recognition of DNA was mediated mainly by amino acids of the target recognition domain, although some amino acids (loop 80–88) of the catalytic domain may also contribute to DNA recognition. These interactions involved direct contacts between M.HgiDII and DNA, as well as indirect contacts through water bridges. Additionally, analysis of sequence alignments with closely related MTases helped us to identify a motif in the TRD of M.HgiDII that may be relevant to specific DNA recognition.