The type I restriction endonuclease EcoR124I, couples ATP hydrolysis to bidirectional DNA translocation

Type I restriction endonuclease holoenzymes contain methylase (M), restriction (R) and specificity (S) subunits, present in an M2:R2:S1 stoichiometry. These enzymes bind to specific DNA sequences and translocate dsDNA in an ATP-dependent manner toward the holoenzyme anchored at the recognition sequence. Once translocation is impeded, DNA restriction, which functions to protect the host cell from invading DNA, takes place. Translocation and DNA cleavage are afforded by the two diametrically opposed R-subunits. To gain insight into the mechanism of translocation, a detailed characterization of the ATPase activity of EcoR124I was done. Results show that following recognition sequence binding, ATP hydrolysis-coupled, bidirectional DNA translocation by EcoR124I ensues, with the R-subunits transiently disengaging, on average, every 515 bp. Macroscopic processivity of 2031(+/-184)bp is maintained, as the R-subunits remain in close proximity to the DNA through association with the methyltransferase. Transient uncoupling of ATP hydrolysis from translocation results in 3.1(+/-0.4) ATP molecules being hydrolyzed per base-pair translocated per R-subunit. This is the first clear demonstration of the coupling of ATP hydrolysis to dsDNA translocation, albeit inefficient. Once translocation is impeded on supercoiled DNA, the DNA is cleaved. DNA cleavage inactivates the EcoR124I holoenzyme partially and reversibly, which explains the stoichiometric behaviour of type I restriction enzymes. Inactivated holoenzyme remains bound to the DNA at the recognition sequence and immediately releases the nascent ends. The release of nascent ends was demonstrated using a novel, fluorescence-based, real-time assay that takes advantage of the ability of the Escherichia coli RecBCD enzyme to unwind restricted dsDNA. The resulting unwinding of EcoR124I-restricted DNA by RecBCD reveals coordination between the restriction-modification and recombination systems that functions to destroy invading DNA efficiently. In addition, we demonstrate the displacement of EcoR124I following DNA cleavage by the translocating RecBCD enzyme, resulting in the restoration of catalytic function to EcoR124I.     

Subunit assembly for DNA cleavage by restriction endonuclease SgrAI

The SgrAI endonuclease usually cleaves DNA with two recognition sites more rapidly than DNA with one site, often converting the former directly to the products cut at both sites. In this respect, SgrAI acts like the tetrameric restriction enzymes that bind two copies of their target sites before cleaving both sites concertedly. However, by analytical ultracentrifugation, SgrAI is a dimer in solution though it aggregates to high molecular mass species when bound to its specific DNA sequence. Its reaction kinetics indicate that it uses different mechanisms to cleave DNA with one and with two SgrAI sites. It cleaves the one-site DNA in the style of a dimeric restriction enzyme acting at an individual site, mediating neither interactions in trans, as seen with the tetrameric enzymes, nor subunit associations, as seen with the monomeric enzymes. In contrast, its optimal reaction on DNA with two sites involves an association of protein subunits: two dimers bound to sites in cis may associate to form a tetramer that has enhanced activity, which then cleaves both sites concurrently. The mode of action of SgrAI differs from all restriction enzymes characterised previously, so this study extends the range of mechanisms known for restriction endonucleases.     

Site-specific DNA-nicking mutants of the heterodimeric restriction endonuclease R.BbvCI

The restriction enzyme R.BbvCI cleaves duplex DNA within a seven base-pair asymmetric recognition sequence, thus: CCTCAGC/GCTGAGG-->CC--TCAGC/GC--TGAGG. We show that R.BbvCI comprises two different subunits, R(1) and R(2); that each subunit contains a catalytic site for DNA strand hydrolysis; and that these sites act independently and strand-specifically. In turn, each catalytic site was inactivated by mutagenesis to form dimeric enzymes in which only one site remained functional. The altered enzymes hydrolyzed just one strand of the recognition sequence, nicking the DNA rather than cleaving it. Enzymes in which the catalytic site in the R(1) subunit remained functional nicked the bottom strand of the sequence, producing CCTCAGC/GC--TGAGG, while those in which the catalytic site in the R(2) subunit remained functional nicked the top strand, producing CC--TCAGC/GCTGAGG. These DNA-nicking enzymes could prove useful for investigation of DNA repair, recombination, and replication, and for laboratory procedures that initiate from nicks, such as DNA degradation, synthesis, and amplification.     

限制性内切酶Mlu I蛋白及其硒代衍生物的制备

[背景]限制性内切酶Mlu I是一种常用的工具酶,在分子生物学领域发挥着重要的作用,其三维结构尚未被解析.[目的]在大肠杆菌中克隆表达、纯化重组Mlu I蛋白及其硒代蛋白,并进行结晶条件的研究.[方法]构建重组表达载体pET28b-Mlu I,在大肠杆菌BL21(DE3)pLysS中诱导表达,利用亲和层析和凝胶过滤层析...     

Polymorphism of mitochondrial DNA in pigs based on restriction endonuclease cleavage patterns

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of pigs and Japanese wild boars were analyzed using 17 enzymes which recognize six nucleotides. The map of cleavage sites was made by double-digestion methods. Polymophism of mtDNA was detected in the digestion by BglII, EcoRV, ScaI, and StuI. The restriction cleavage patterns were identical among the breeds of Landrace, Hampshire, Duroc I, and Large White I (A type). The patterns of Large White II were the same as those of Japanese wild boars (B type). A difference between the A type and the B type of mtDNA was found in the case of three restriction enzymes, BglII, ScaI, and StuI, and the nucleotide alterations between them were estimated as more than six. On the other hand, a difference between mtDNA from almost all pigs and mtDNA from Duroc II was detected using EcoRV. We suggest that the difference of mtDNA between the A type and the B type of mtDNA could result from the different origin of boars, that is, whether they were of European or Asian origin.     

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg²⁺ ions, EcoRV is able to cleave the DNA but not in presence of Ca²⁺, although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg²⁺(A)-EcoRV-DNA complex compared to Ca²⁺(S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca²⁺(S)-EcoRV-DNA compared to Mg²⁺(A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca²⁺(S) bound complex than in Mg²⁺(A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.     

The different banding patterns produced by restriction endonuclease digestion in mitotic chromosomes of the American and European eel

The detection of three classes of C-heterochromatin by in situ restriction endonuclease digestion allowed a karyotype differentiation between the American and the European eel.     

Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine population of native cattle, indicating the existence of two separate types of mtDNA. These two types of mtDNA are very different from each other, at the level of subspecies. Since the native Philippine cattle are considered to represent an admixture of European and Indian cattle, the two types of mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and the nucleotide substitutions at some of the sites have also been estimated.     

Modes of DNA cleavage by the EcoRV restriction endonuclease

The mechanism of action of the EcoRV restriction endonuclease at its single recognition site on the plasmid pAT153 was analyzed by kinetic methods. In reactions at pH 7.5, close to the optimum for this enzyme, both strands of the DNA were cut in a single concerted reaction: DNA cut in only one strand of the duplex was neither liberated from the enzyme during the catalytic turnover nor accumulated as a steady-state intermediate. In contrast, reactions at pH 6.0 involved the sequential cutting of the two strands of the DNA. Under these conditions, DNA cut in a single strand was an obligatory intermediate in the reaction pathway and a fraction of the nicked DNA dissociated from the enzyme during the turnover. The different reaction profiles are shown to be consistent with a single mechanism in which the kinetic activity of each subunit of the dimeric protein is governed by its affinity for Mg2+ ions. At pH 7.5, Mg2+ is bound to both subunits of the dimer for virtually the complete period of the catalytic turnover, while at pH 6.0 Mg2+ is bound transiently to one subunit at a time. The kinetics of the EcoRV nuclease were unaffected by DNA supercoiling.     

Selective inhibition of restriction endonuclease cleavage by DNA intercalators

The preferred dye binding sites and the microenvironment of known nucleotide sequences within mitochondrial and plasmid pBR322 DNA was probed in a gross fashion with restriction endonucleases. The intercalating dyes, ethidium bromide and propidium iodide, do not inhibit a given restriction endonuclease equally at all of the restriction sites within a DNA molecule. The selective inhibition may be explained, in part, by the potential B to Z conformation transition of DNA flanking the restriction site and by preferred dye binding sites. Propidium iodide was found to be a more potent inhibitor than ethidium bromide and the inhibition is independent of the type of cut made by the enzyme.     

Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease

According to the current paradigm type IIE restriction endonucleases are homodimeric proteins that simultaneously bind to two recognition sites but cleave DNA at only one site per turnover: the other site acts as an allosteric locus, activating the enzyme to cleave DNA at the first. Structural and biochemical analysis of the archetypal type IIE restriction enzyme EcoRII suggests that it has three possible DNA binding interfaces enabling simultaneous binding of three recognition sites. To test if putative synapsis of three binding sites has any functional significance, we have studied EcoRII cleavage of plasmids containing a single, two and three recognition sites under both single turnover and steady state conditions. EcoRII displays distinct reaction patterns on different substrates: (i) it shows virtually no activity on a single site plasmid; (ii) it yields open-circular DNA form nicked at one strand as an obligatory intermediate acting on a two-site plasmid; (iii) it cleaves concertedly both DNA strands at a single site during a single turnover on a three site plasmid to yield linear DNA. Cognate oligonucleotide added in trans increases the reaction velocity and changes the reaction pattern for the EcoRII cleavage of one and two-site plasmids but has little effect on the three-site plasmid. Taken together the data indicate that EcoRII requires simultaneous binding of three rather than two recognition sites in cis to achieve concerted DNA cleavage at a single site. We show that the orthodox type IIP enzyme PspGI which is an isoschisomer of EcoRII, cleaves different plasmid substrates with equal rates. Data provided here indicate that type IIE restriction enzymes EcoRII and NaeI follow different mechanisms. We propose that other type IIE restriction enzymes may employ the mechanism suggested here for EcoRII.     

The time-resolved kinetics of superhelical DNA cleavage by BamHI restriction endonuclease

The kinetics of the cleavage of superhelical plasmid DNA (pBR322) by the restriction endonuclease, BamHI, have been analyzed in terms a compartmental model consistent with the chemistry first proposed by Rubin and Modrich (Rubin, R. A., and Modrich, P. (1978) Nucleic Acids Res. 5, 2991-2997) for analysis of the kinetics of the restriction endonuclease, EcoRI. The model was defined in terms of two compartments representing DNA substrate (bound and free), two compartments representing nicked intermediate (bound and free), one compartment representing linear product, and one compartment for free enzyme. A simultaneous analysis of concentration changes over time of the three DNA forms (superhelical, nicked, and linear) at six different enzyme concentrations was undertaken employing this compartmental model using SAAM (Simulation Analysis And Modeling) software. Results showed that rate constants characterizing the association of enzyme with superhelical DNA (6.0 x 10(5) M-1 s-1) and nicked DNA (2.8 x 10(5) M-1 s-1) were similar in magnitude and rate constants characterizing cleavage of the first (1.2 x 10(-2) s-1) and second phosphodiester bonds (3.1 x 10(-2) s-1) were also similar. The analysis yields a kinetically determined equilibrium constant of 12.9 nM for the dissociation of nicked intermediate from the enzyme. The rate constant describing the release of the nicked intermediate from the enzyme has a value of 3.7 x 10(-3) s-1. By comparing the value of this release rate constant to the value of the constant describing the second cleavage event, it can be determined that only 10% of the nicked intermediate bound to the enzyme is released as free nicked DNA and that 90% of the nicked intermediate is processed to the linear form without being released. Hence, most of the DNA is cleaved as the result of a single enzyme-DNA recognition event. No steady state assumptions were made in the analysis. The approach was to directly solve the differential equations which described the kinetic processes using an interactive method. This study demonstrates the usefulness of this approach for the analysis of kinetics of protein-DNA interactions for the restriction endonucleases.     

Processive cleavage of concatemer DNA duplexes by Eco RII restriction endonuclease

Eco RII restriction endonuclease cleaves synthetic DNA-duplexes in which the recognition sites of this enzyme (5..CC _T ^A GG...) are repeated every 9 base pairs with the alternating orientation of the central AT pair. It operates in a processive mode, i.e. the bound enzyme molecule slides along the substrate toward neighboring recognition sites. Nona-nucleotides are the main products of the cleavage. The data obtained point to the capability of Eco RII endonuclease to recognize and cleave the substrate under both possible orientations of the central AT-pair of the recognition site with respect to the bound enzyme molecule. These data also show the close similarity of DNA structures in a complex with theenzyme and without.     

Site-dependent cleavage of pBR322 DNA by restriction endonuclease HinfI.

Cleavage of pBR322 DNA I by the restriction endonuclease HinfI is preferentially inhibited at specific HinfI cleavage sites. These sites in pBR322 DNA I have been identified and ordered with respect to the frequency with which they are cleaved. The HinfI site most resistant to cleavage in pBR322 DNA I is unique in that runs of G-C base pairs are immediately adjacent on both sites. Two differently permuted linear (DNA III) species were produced by cleavage with two different restriction endonucleases, PstI and AvaI. Only one of these linear molecules, that produced by PstI, exhibits the same preferential cleavage pattern as DNA I. The second linear species, that arising from AvaI digestion, shows pronounced relative inhibition of cleavage at the HinfI sites nearest the ends of the molecule (100 to 120 base pairs away, respectively). This result suggest that proximity to the termini of a linear DNA molecule might also influence preferential cleavage. The possibility of formation of stem-loop structures does not appear to influence preferential cleavage by HinfI.     

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein,a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

Mycobacterium leprae recA harbors an in‐frame insertion sequence that encodes an intein homing endonuclease (PI‐MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG‐type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI‐MleI is distinctive from other members of the family of LAGLIDADG‐type HEases for its modular structure with functionally separable domains for DNA‐binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI‐MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active‐site residues essential for DNA target site recognition and double‐stranded DNA cleavage, we performed site‐directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild‐type PI‐MleI and its variants disclosed that the two amino acid residues, Asp¹²² (in Block C) and Asp¹⁹³ (in functional Block E), are crucial to the double‐stranded DNA endonuclease activity, whereas Asp²¹⁸ (in pseudo‐Block E) is not. However, despite the reduced catalytic activity, the PI‐MleI variants, like the wild‐type PI‐MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA‐binding affinities, but abolished the double‐stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double‐stranded DNA cleavage activity, compared with the wild‐type PI‐MleI. These results provide compelling evidence that Asp¹²² and Asp¹⁹³ in DOD motif I and II, respectively, are bona fide active‐site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.     

The inverted terminal repetition of the DNA of weakly oncogenic adenovirus type 7

By use of the chemical modification technique of Maxam and Gilbert (1977), the first 180 base pairs at both termini of the human adenovirus 7 genome have been determined. The results show that adenovirus 7 DNA contains a perfect inverted terminal repetition of 136 base pairs.     

Scanning force microscopy of DNA translocation by the Type III restriction enzyme EcoP15I

Type III restriction enzymes are multifunctional heterooligomeric enzymes that cleave DNA at a fixed position downstream of a non-symmetric recognition site. For effective DNA cleavage these restriction enzymes need the presence of two unmethylated, inversely oriented recognition sites in the DNA molecule. DNA cleavage was proposed to result from ATP-dependent DNA translocation, which is expected to induce DNA loop formation, and collision of two enzyme-DNA complexes. We used scanning force microscopy to visualise the protein interaction with linear DNA molecules containing two EcoP15I recognition sites in inverse orientation. In the presence of the cofactors ATP and Mg(2+), EcoP15I molecules were shown to bind specifically to the recognition sites and to form DNA loop structures. One of the origins of the protein-clipped DNA loops was shown to be located at an EcoP15I recognition site, the other origin had an unspecific position in between the two EcoP15I recognition sites. The data demonstrate for the first time DNA translocation by the Type III restriction enzyme EcoP15I using scanning force microscopy. Moreover, our study revealed differences in the DNA-translocation processes mediated by Type I and Type III restriction enzymes.     

DNA structural polymorphism modulates the kinetics of superhelical DNA cleavage by BamHI restriction endonuclease

A compartmental model developed by Hensley (Hensley, P., Nardone, G., Chirikjian, J.G., and Wastney, M. E., (1990) J. Biol. Chem. 265, 15300-15307) for analysis of the time courses of the cleavage of superhelical DNA substrates by the restriction endonuclease, BamHI, has been used to quantify the effects of changes in temperature, ionic strength, superhelical density, and the DNA substrate on the binding and strand cleavage processes. Studies reported here indicate that changes in topology may be introduced into the DNA substrate solely as a result of the plasmid preparation process and in the absence of covalent bond cleavage and ligation. These changes in topology have qualitatively different effects on the kinetics than those promoted by changes in the superhelical density. The former are removed by briefly warming the DNA prior to assay, suggesting that they are only kinetically stable, while the latter changes are not affected by heating. Increasing the [NaCl] from 0.01 M to 0.1 M increases the overall rate of plasmid cleavage by increasing both the rates of cleavage and enzyme DNA association. To describe the decrease in the overall cleavage rate observed in 0.15 M NaCl, an ionic strength-dependent rate-determining structural transition in the DNA substrate was incorporated into the model. The largest changes in the rate of the cleavage process resulted from changes in the DNA substrate. For the SV40 substrate compared to pBR322, the rate constants describing the two association processes and the first bond cleavage event were increased 6- to 7-fold. The rate of the second bond cleavage process was not affected. These changes may be due to differences in the flanking sequences.     

S-adenosyl methionine prevents promiscuous DNA cleavage by the EcoP1I type III restriction enzyme

DNA cleavage by the type III restriction endonuclease EcoP1I was analysed on circular and catenane DNA in a variety of buffers with different salts. In the presence of the cofactor S-adenosyl methionine (AdoMet), and irrespective of buffer, only substrates with two EcoP1I sites in inverted repeat were susceptible to cleavage. Maximal activity was achieved at a Res2Mod2 to site ratio of approximately 1:1 yet resulted in cleavage at only one of the two sites. In contrast, the outcome of reactions in the absence of AdoMet was dependent upon the identity of the monovalent buffer components, in particular the identity of the cation. With Na+, cleavage was observed only on substrates with two sites in inverted repeat at elevated enzyme to site ratios (>15:1). However, with K+ every substrate tested was susceptible to cleavage above an enzyme to site ratio of approximately 3:1, including a DNA molecule with two directly repeated sites and even a DNA molecule with a single site. Above an enzyme to site ratio of 2:1, substrates with two sites in inverted repeat were cleaved at both cognate sites. The rates of cleavage suggested two separate events: a fast primary reaction for the first cleavage of a pair of inverted sites; and an order-of-magnitude slower secondary reaction for the second cleavage of the pair or for the first cleavage of all other site combinations. EcoP1I enzymes mutated in either the ATPase or nuclease motifs did not produce the secondary cleavage reactions. Thus, AdoMet appears to play a dual role in type III endonuclease reactions: Firstly, as an allosteric activator, promoting DNA association; and secondly, as a "specificity factor", ensuring that cleavage occurs only when two endonucleases bind two recognition sites in a designated orientation. However, given the right conditions, AdoMet is not strictly required for DNA cleavage by a type III enzyme.