UV- and CD-spectra of restriction endonuclease EcoRII and DNA-methylase EcoRII

UV- and CD-spectra of homogeneous enzymes have been measured. Extinction coefficients estimated from the UV-spectra are 0.97 for restriction endonuclease EcoRII at 279.5 nm and 1.17 for DNA-methylase EcoRII at 279 nm. As it follows from the CD spectra, both enzymes have a well developed tertiary structure and a highly ordered secondary structure, which consists of 22% alpha-helices, 64% beta-structure and 9% bends for REcoRII and of 44% alpha-helices, 48% beta-structure and 4% bends for MEcoRII. Restriction endonuclease denatures at 50 degrees C, while DNA-methylase denatures at 45 degrees C, with partial reversibility upon cooling.     

Asymmetric photocross-linking pattern of restriction endonuclease EcoRII to the DNA recognition sequence

The EcoRII homodimer engages two of its recognition sequences (5'-CCWGG) simultaneously and is therefore a type IIE restriction endonuclease. To identify the amino acids of EcoRII that interact specifically with the recognition sequence, we photocross-linked EcoRII with oligonucleotide substrates that contained only one recognition sequence for EcoRII. In this recognition sequence, we substituted either 5-iododeoxycytidine for each C or 5-iododeoxyuridine for A, G, or T. These iodo-pyrimidine bases were excited using a UV laser to result in covalent cross-linking products. The yield of EcoRII photocross-linked to the 5'-C of the 5'-CCAGG strand of the recognition sequence was 45%. However, we could not photocross-link EcoRII to the 5'-C of the 5'-CCTGG strand. Thus, the contact of EcoRII to the bases of the recognition sequence appears to be asymmetric, unlike that expected for most type II restriction endonucleases. Tryptic digestion of free and of cross-linked EcoRII, followed by high performance liquid chromatography (HPLC) separation of the individual peptides and Edman degradation, identified amino acids 25-49 of EcoRII as the cross-linking peptide. Mutational analysis of the electron-rich amino acids His(36) and Tyr(41) of this peptide indicates that Tyr(41) is the amino acid involved in the cross-link and that it therefore contributes to specific DNA recognition by EcoRII.     

Nucleotide sequence of the EcoRII restriction endonuclease gene

The nucleotide sequence of a 1394 basepair (bp) DNA fragment containing the EcoRII restriction endonuclease (R.EcoRII) gene was determined. The endonuclease gene is 1206 bp in length (predicted 402 amino acids (aa) and Mr = 45 178) and is separated by 33 bp from the EcoRII modification methylase (M.EcoRII) gene. The EcoRII restriction-modification system has a tail-to-tail organization of the two genes.     

Purification of restriction endonuclease EcoRII using monoclonal antibodies

Monoclonal antibodies against EcoRII endonuclease were obtained after immunization of two BALB/c mice with a homogeneous enzyme prepared by conventional methods. IgG from ascitic fluid was purified and coupled to CNBr-activated Sepharose 4B to give a specific column used to isolate EcoRII endonuclease. The isolated EcoRII endonuclease produced a single band during SDS gel electrophoresis.     

Isolation, purification and properties of restriction endonuclease EcoRII

The restriction endonuclease Eco RII was isolated and purified to homogeneity. The isolation procedure involved the use of the E. coli strain B834/pSK323, containing the recombinant plasmide pSK323 which provides for the oversynthesis of Eco RII enzymes. Data from gel filtration and Na-DS electrophoresis suggest that the restriction endonuclease Eco RII is a protein made up of two subunits, each with molecular weight of 44 000.     

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.     

Preparation of DNA-duplexes, containing internucleotide thiophosphate groups in various positions of the recognition site for the EcoRII restriction endonuclease

Twenty-four 12-mer DNA duplexes, each containing a chiral phosphorothioate group successively replacing one of the internucleotide phosphate groups either in the EcoRII recognition site (5'CCA/TGG) or near to it, were obtained for studying the interaction of the restriction endonuclease EcoRII with internucleotide DNA phosphates. Twelve of the 12-mer oligonucleotides were synthesized as Rp and Sp diastereomeric mixtures. Six of them were separated by reversed-phase HPLC using various buffers. Homogeneous diastereomers of the other oligonucleotides were obtained by enzymatic ligation of the Rp and Sp diastereomers of 5- to 7-mer oligonucleotides preliminarily separated by HPLC with the corresponding short oligonucleotides on a complementary DNA template. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites.

EcoRII is unusual among type II restriction enzymes in that, while it cleaves substrates such as pBR322 and bacteriophage lambda that contain several recognition sites for the enzyme efficiently, substrates such as the genomes of bacteriophages T3 and T7 which contain a small number of recognition sites are cut poorly by it. Interestingly, pBR322, or a short DNA duplex containing a single site for the enzyme, can activate the enzyme to cleave resistant substrates. We show here that, at low concentrations, activator short duplexes are themselves cleaved poorly by the enzyme. Further, the reaction shows substrate cooperativity, and at high concentrations, the duplexes are both activators and good substrates for the enzyme. This supports the model that the activation of EcoRII involves binding of more than one DNA molecule and provides a simple system to study the mechanism of activation. Using a gel mobility shift assay, we show that the enzyme forms sequence-specific, methylation-sensitive complexes with the duplexes in the absence of activating DNA. Therefore, resistance of the short duplexes to the enzyme at low concentrations cannot be due to an inability of the enzyme to bind the duplexes. Interestingly, these complexes are stable in the presence of Mg2+, the cofactor for the enzyme, and the complexes obtained in the presence of Mg2+ do not contain DNA that is cleaved by the enzyme. The inefficient step in the action of EcoRII on resistant substrates must occur subsequent to initial substrate binding and it is this step that the activating DNA must regulate.     

Creation of a type IIS restriction endonuclease with a long recognition sequence

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.     

Interaction of restriction and modification enzyme EcoRII with synthetic DNA fragments. VII. The study of complex-formation of endonuclease EcoRII with substrates containing natural and modified recognition sites

As shown by a nitrocellulose filter binding assay, in the absence of Mg2+ EcoRII restriction endonuclease binds specifically to a set of synthetic concatemer DNA duplexes of varying chain length, containing natural and modified recognition sites of this enzyme. The binding of the substrates with the central AT, TT or AA-pair in the recognition site decreases at AT greater than TT much greater than AA. Substitution of the pyrophosphate bond at the cleavage site for the phosphodiester or phosphoramide bond produces little influence on the stability of the complexes. The affinity of the enzyme for nonspecific sites is two orders of magnitude less than that for the specific EcoRII sequences. Equilibrium association constant for a substrate with one recognition site is 3.9 X 10(8) M-1. Addition of Mg2+ leads to the destabilization of the EcoRII endonuclease complex with DNA duplex, containing pyrophosphate bonds. The dissociation rate constants and the lifetime of the EcoRII endonuclease--synthetic substrates complexes have been determined.     

Activation of restriction endonuclease EcoRII does not depend on the cleavage of stimulator DNA.

The restriction endonuclease EcoRII is unable to cleave DNA molecules when recognition sites are very far apart. The enzyme, however can be activated in the presence of DNA molecules with a high frequency of EcoRII sites or by oligonucleotides containing recognition sites: Addition of the activator molecules stimulates cleavage of the refractory substrate. We now show that endonucleolysis of the stimulator molecules is not a necessary prerequisite of enzyme activation. A total EcoRII digest of pBR322 DNA or oligonucleotide duplexes with simulated EcoRII ends (containing the 5' phosphate group), as well as oligonucleotide duplexes containing modified bases within the EcoRII site, making them resistant to cleavage, are all capable of enzyme activation. For activation EcoRII requires the interaction with at least two recognition sites. The two sites may be on the same DNA molecule, on different oligonucleotide duplexes, or on one DNA molecule and one oligonucleotide duplex. The efficiency of functional intramolecular cooperation decreases with increasing distance between the sites. Intermolecular site interaction is inversely related to the size of the stimulator oligonucleotide duplex. The data are in agreement with a model whereby EcoRII simultaneously interacts with two recognition sites in the active complex, but cleavage of the site serving as an allosteric activator is not necessary.     

Mechanism of the interaction of EcoRII restriction endonuclease with two recognition sites. Probing of modified DNA duplexes as activators of the enzyme.

The efficiency of cleavage of DNA duplexes with single EcoRII recognition sites by the EcoRII restriction endonuclease decreases with increasing substrate length. DNA duplexes of more than 215 bp are not effectively cleaved by this enzyme. Acceleration of the hydrolysis of long single-site substrates by EcoRII is observed in the presence of 11-14-bp substrates. The stimulation of hydrolysis depends on the length and concentration of the second substrate. To study the mechanism of EcoRII endonuclease stimulation, DNA duplexes with base analogs and modified internucleotide phosphate groups in the EcoRII site have been investigated as activators. These modified duplexes are cleaved by EcoRII enzyme with different efficiencies or are not cleaved at all. It has been discovered that the resistance of some of them can be overcome by incubation with a susceptible canonical substrate. The acceleration of cleavage of long single-site substrates depends on the type of modification of the activator. The modified DNA duplexes can activate EcoRII catalyzed hydrolysis if they can be cleaved by EcoRII themselves or in the presence of the second canonical substrate. It has been demonstrated that EcoRII endonuclease interacts in a cooperative way with two recognition sites in DNA. The cleavage of one of the recognition sites depends on the cleavage of the other. We suggest that the activator is not an allosteric effector but acts as a second substrate.     

A study of the Asp110-Glu112 region of EcoRII restriction endonuclease by site-directed mutagenesis

Site-directed mutagenesis of the ecoRII gene has been used to search for the active site of the EcoRII restriction endonuclease. Plasmids with point mutations in ecoRII gene resulting in substitutions of amino acid residues in the Asp110-Glu112 region of the EcoRII endonuclease (Asp110 --> Lys, Asn, Thr, Val, or Ile; Pro111 --> Arg, His, Ala, or Leu; Glu112 --> Lys, Gln, or Asp) have been constructed. When expressed in E. coli, all these plasmids displayed EcoRII endonuclease activity. We also constructed a plasmid containing a mutant ecoRII gene with deletion of the sequence coding the Gln109-Pro111 region of the protein. This mutant protein had no EcoRII endonuclease activity. The data suggest that Asp110, Pro111, and Glu112 residues do not participate in the formation of the EcoRII active site. However, this region seems to be relevant for the formation of the tertiary structure of the EcoRII endonuclease.     

Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage

EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-18 bases. Single amino acid replacement of Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val(258) is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding.     

A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.     

Specific DNA recognition by the type II restriction endonuclease MunI: the effect of pH.

To investigate the effect of pH on sequence-specific binding, a thermodynamic characterization of the interaction of the protein MunI with a specific, and a nonspecific, oligonucleotide was performed. MunI is a type II restriction endonuclease which is able to bind specifically, but loses its enzymatic activity in the absence of magnesium ions. Comparison of the specific and nonspecific interactions at 10 and 25 degrees C shows that the latter is accompanied by a small change in enthalpy, and a negligible change in constant pressure heat capacity. On going through the pH range 5.75-9.0 at 25 degrees C, the affinity of specific complex formation is reduced by 20-fold. The interaction is accompanied by the protonation of groups assumed to be on the protein. Based on the simplest model that will fit the data, two distinct protonation events are observed. At low pH, two groups per protein molecule undergo protonation with a pK(a) of 6.0 and 6.9 in the free and bound forms, respectively. At high pH, a further independent protonation occurs involving two groups with pK(a) values of 8.9 and approximately 10.7 in the free and bound forms, respectively. The change in heat capacity ranges from -2.7 to -1.7 kJ mol(-1) K(-1) in going from pH 6.5 to 8.5. This range of variation of change in heat capacity can be accounted for by the effects of protonation of the interacting molecules. The change in heat capacity, calculated from surface area burial using a previously established relationship (1.15 kJ mol(-1) K(-1)), does not correlate well with the experimentally determined values.     

Effects of benzo[a]pyrene-deoxyguanosine lesions on DNA methylation catalyzed by EcoRII DNA methyltransferase and on DNA cleavage effected by EcoRII restriction endonuclease

DNA methylation is an important cellular mechanism for controlling gene expression. Whereas the mutagenic properties of many DNA adducts, e.g., those arising from polycyclic aromatic hydrocarbons, have been widely studied, little is known about their influence on DNA methylation. We have constructed site-specifically modified 18-mer oligodeoxynucleotide duplexes containing a pair of stereoisomeric adducts derived from a benzo[a]pyrene-derived diol epoxide [(+)- and (-)-r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, or B[a]PDE] bound to the exocyclic amino group of guanine. The adducts, either (+)- or (-)-trans-anti-B[a]P-N(2)-dG (G*), positioned either at the 5'-side or the 3'-side deoxyguanosine residue in the recognition sequence of EcoRII restriction-modification enzymes (5'-...CCA/TGG...) were incorporated into 18-mer oligodeoxynucleotide duplexes. The effects of these lesions on complex formation and the catalytic activity of the EcoRII DNA methyltransferase (M.EcoRII) and EcoRII restriction endonuclease (R.EcoRII) were investigated. The M.EcoRII catalyzes the transfer of a methyl group to the C5 position of the 3'-side cytosine of each strand of the recognition sequence, whereas R.EcoRII catalyzes cleavage of both strands. The binding of R.EcoRII to the oligodeoxynucleotide duplexes and the catalytic cleavage were completely abolished when G was positioned at the 3'-side dG position (5'-...CCTGG*...). When G* was at the 5'-side dG position, binding was moderately diminished, but cleavage was completely blocked. In the case of M.EcoRII, binding is diminished by factors of 5-30 but the catalytic activity was either abolished or reduced 4-80-fold when the adducts were located at either position. Somewhat smaller effects were observed with hemimethylated oligodeoxynucleotide duplexes. These findings suggest that epigenetic effects, in addition to genotoxic effects, need to be considered in chemical carcinogenesis initiated by B[a]PDE, since the inhibition of methylation may allow the expression of genes that promote tumor development.