Change in the cleavage site of synthetic substrates of SsoII restriction endonucleases upon introduction of non-nucleotide inserts in the recognition segment]

The cleavage of synthetic DNA duplexes containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) residues instead of one of dG residues or one of the nucleosides of the central base pair of the recognition site by SsoII restriction endonuclease (decreases CCNGG) has been studied. It is found that the non-nucleotide insertions (except for glG) result in a change of the SsoII cleavage site and an increase of the efficiency of the cleavage. The novel noncanonical cleavage occurs at the phosphodiester bond adjoining the non-nucleotide insert from the 5'-end.     

Cleavage by restriction endonucleases MvaI and EcoRII of substrates modified in amino groups of heterocyclic bases

14-membered DNA-duplexes containing modified nucleoside residues, viz 4-N-methyldeoxycytidine (m4dC), 6-N-methyldeoxyadenosine (m6dA) or deoxyinosine (dI), in only one strand of the recognition site (CCA/TGG) of MvaI and EcoRII endonucleases were synthesized. It was shown that MvaI and EcoRII endonucleases interact with the exocyclic amino groups of the external dC residues and of the central dA residue of the recognition site exposed into the DNA major groove. These endonucleases which are isochizomers were found to possess different mechanisms of substrate cleavage. The ability of MvaI endonuclease to hydrolyze only unmodified strand of methylated duplexes allows one to make site-directed single-strand nicks in double-stranded DNA. Elimination of the 2-NH2-group located in the minor groove of DNA by substituting dI for dG had little, if any, effect on the hydrolytic activity of EcoRII and MvaI endonucleases.     

Cleavage of concatamer-type substrates by restriction endonucleases MVA1 and SSO1I

The interaction of enzymes SsoII (decreases CCNGG) and MvaI (CC decreases A/TGG) with concatemeric DNA duplexes used earlier to study EcoRII (decreases CCA/TGG) TGG was investigated with a view of elucidating the general principles of the restriction endonuclease function. A pattern common for all the three enzymes was observed with DNA duplexes containing AA or TT pairs in the central position of the recognition site. The AA pair blocks or substantially hinders the endonuclease action, whereas the TT pair is either less inhibitory or altogether inert. SsoII, similar to EcoRII was able to processively cleave the concatemeric substrates and to interact with (or to be close to) the hydrogen in the 5th position of the outer dC residue of the recognition site. MvaI was found to differ from EcoRII in the way they recognize and cleave the same nucleotide sequence. The substrate-bound MvaI molecule is incapable of linear diffusion along the DNA. Effective hydrolysis of dU- and m5dC-containing polymers rules out the participation of hydrophobic contacts of the enzyme with the methyl group of the dT residue and with the 5th hydrogen of the outer dC residue of the recognition site in DNA-protein interactions.     

PspGI,a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes

We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.     

Peculiarities of recognition of CCA/TGG sequences in DNA by restriction endonucleases MvaI and EcoRII.

To elucidate the mechanism of action of restriction endonucleases MvaI and EcoRII a study was made of their interaction with a set of synthetic substrates in which the heterocyclic bases or the sugar-phosphate backbone had been modified; individual nucleotide residues had been removed or replaced with hydrocarbon bridges, and mismatched base pairs had been introduced. The groups of atoms in the heterocyclic bases and the phosphates in the recognition site that produce the most significant influence on the functioning of endonucleases MvaI and EcoRII were discerned. Profound differences were found in the functioning of the MvaI and EcoRII neoschizomers. The catalytic activity of EcoRII is significantly affected by any alteration in the recognition site structure and conformation, with a modification in one strand of the substrate causing the same decrease in the hydrolysis rate of both strands. Endonuclease MvaI is tolerant to a number of structural abnormalities; the latter sometimes affect only hydrolysis of one strand of the recognition site. The enzyme can preferentially cleave one of the substrate strands. Mismatched base pairs retard and sometimes block the hydrolysis. The effect depends on the particular enzyme, mismatch and its location.     

Specificity changes in the evolution of type II restriction endonucleases: a biochemical and bioinformatic analysis of restriction enzymes that recognize unrelated sequences

How restriction enzymes with their different specificities and mode of cleavage evolved has been a long standing question in evolutionary biology. We have recently shown that several Type II restriction endonucleases, namely SsoII (downward arrow CCNGG), PspGI (downward arrow CCWGG), Eco-RII (downward arrow CCWGG), NgoMIV (G downward arrow CCGGC), and Cfr10I (R downward arrow CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of approximately 70 amino acid residues with MboI, a Type II restriction endonuclease from Moraxella bovis (Pingoud, V., Conzelmann, C., Kinzebach, S., Sudina, A., Metelev, V., Kubareva, E., Bujnicki, J. M., Lurz, R., Luder, G., Xu, S. Y., and Pingoud, A. (2003) J. Mol. Biol. 329, 913-929). Nevertheless, MboI has a dissimilar DNA specificity (downward arrow GATC) compared with these enzymes. In this study, we characterize MboI in detail to determine whether it utilizes a mechanism of DNA recognition similar to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. Mutational analyses and photocross-linking experiments demonstrate that MboI exploits the stretch of approximately 70 amino acids for DNA recognition and cleavage. It is therefore likely that MboI shares a common evolutionary origin with SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. This is the first example of a relatively close evolutionary link between Type II restriction enzymes of widely different specificities.     

Evolutionary relationship between different subgroups of restriction endonucleases

The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.     

Identification of base-specific contacts in protein-DNA complexes by photocrosslinking and mass spectrometry: a case study using the restriction endonuclease SsoII

Specific protein-nucleic acid interactions are of paramount importance for the propagation, maintenance and expression of genetic information. Restriction endonucleases serve as model systems to study the mechanisms of DNA recognition by proteins. SsoII is a Type II restriction endonuclease that recognizes the double stranded sequence downward arrow CCNGG and cleaves it in the presence of Mg(2+)-ions, as indicated. SsoII shows sequence similarity over a stretch of approximately 70 amino acid residues with several other restriction endonucleases that recognize a similar sequence as SsoII (Cfr10I, EcoRII, NgoMIV, PspGI). In NgoMIV this stretch is involved in DNA recognition and cleavage, as shown by the crystal structure analysis of an enzyme-product complex. To find out whether the presumptive DNA recognition region in SsoII is indeed in contact with DNA we have photocrosslinked SsoII with an oligodeoxyribonucleotide in which the first guanine of the recognition sequence was replaced by 5-iodouracil. Following digestion by trypsin, the peptide-oligodeoxyribonucleotide conjugate was purified by Fe(3+)-IMAC and then incubated with hydrogen fluoride, which hydrolyzes the oligodeoxyribonucleotide to yield the peptide-deoxyuridine conjugate. The site of photocrosslinking was identified by MALDI-TOF-MS and MALDI-TOF-MS/MS to be Trp189, adjacent to Arg188, which aligns with Arg194 in NgoMIV, involved in recognition of the second guanine in the NgoMIV recognition sequence G downward arrow CCGGC. This result confirms previously published conclusions drawn on the basis of a mutational analysis of SsoII. The methodology that was employed here can be used in principle to identify the DNA binding site of any protein.     

The interaction of DNA duplexes containing 2-aminopurine with restriction endonucleases EcoRII and SsoII.

Oligonucleotides containing 2-aminopurine (2-AP) in place of G or A in the recognition site of EcoRII (CCT/AGG) or SsoII (CCNGG) restriction endonucleases have been synthesized in order to investigate the specific interaction of DNA with these enzymes. Physicochemical properties (CD spectra and melting behaviour) have shown that DNA duplexes containing 2-aminopurine exist largely in a stable B-like form. 2-Aminopurine base paired with cytidine, however, essentially influences the helix structure. The presence of a 2-AP-C mismatch strongly reduces the stability of the duplexes in comparison with the natural double strand, indicated by a biphasic melting behaviour. SsoII restriction endonuclease recognizes and cleaves the modified substrate with a 2-AP-T mismatch in the centre of the recognition site, but it does not cleave the duplexes containing 2-aminopurine in place of inner and outer G, or both. EcoRII restriction endonuclease does not cleave duplexes containing 2-aminopurine at all. The two-substrate mechanism of EcoRII-DNA interaction, however, allows hydrolysis of the duplex containing 2-aminopurine in place of adenine in the presence of the canonical substrate.     

A model of restriction endonuclease MvaI in complex with DNA: a template for interpretation of experimental data and a guide for specificity engineering

R.MvaI is a Type II restriction enzyme (REase), which specifically recognizes the pentanucleotide DNA sequence 5'-CCWGG-3' (W indicates A or T). It belongs to a family of enzymes, which recognize related sequences, including 5'-CCSGG-3' (S indicates G or C) in the case of R.BcnI, or 5'-CCNGG-3' (where N indicates any nucleoside) in the case of R.ScrFI. REases from this family hydrolyze the phosphodiester bond in the DNA between the 2nd and 3rd base in both strands, thereby generating a double strand break with 5'-protruding single nucleotides. So far, no crystal structures of REases with similar cleavage patterns have been solved. Characterization of sequence-structure-function relationships in this family would facilitate understanding of evolution of sequence specificity among REases and could aid in engineering of enzymes with new specificities. However, sequences of R.MvaI or its homologs show no significant similarity to any proteins with known structures, thus precluding straightforward comparative modeling. We used a fold recognition approach to identify a remote relationship between R.MvaI and the structure of DNA repair enzyme MutH, which belongs to the PD-(D/E)XK superfamily together with many other REases. We constructed a homology model of R.MvaI and used it to predict functionally important amino acid residues and the mode of interaction with the DNA. In particular, we predict that only one active site of R.MvaI interacts with the DNA target at a time, and the cleavage of both strands (5'-CCAGG-3' and 5'-CCTGG-3') is achieved by two independent catalytic events. The model is in good agreement with the available experimental data and will serve as a template for further analyses of R.MvaI, R.BcnI, R.ScrFI and other related enzymes.     

Detection of glycosylase, endonuclease and methyltransferase enzyme activities using immobilized oligonucleotides

A novel rapid assay for detection of DNA glycosylase, restriction endonuclease, and DNA methyltransferase enzyme activities is presented. The assay is based on enzyme-dependent label release (in case of glycosylase and endonuclease), or non-release (in case of methyltransferase) into solution from end-labeled DNA immobilized on solid support (CPG or Tenta Gel S-NH2). The assay has been validated for monitoring activity of repair enzyme uracil-DNA glycosylase, restriction endonucleases SsoII, MvaI and EcoRII and (cytosine-5)-DNA methyltransferase SsoII. Two types of labels have been tested and found compatible with the assay: radioactive (32P) and fluorescent (rhodamine B and fluorescein). The enzyme activity is estimated as a ratio of the label released into solution to the total amount of the label. Use of fluorescent labeling facilitates detection while use of solid phase-immobilized substrates facilitates product separation, improved assay sensitivity, and increases throughput of assay. Proposed technique provides an estimate of enzyme activity but not its specific activity. Thus, the assay will most valuable in the applications where rapid estimation of enzyme activity is necessary.     

DNA duplexes containing altered sugar residues as probes of EcoRII and MvaI endonuclease interactions with sugar-phosphate backbone

Oligonucleotides containing 1-(beta-D-2'-deoxy-threo-pentofuranosyl)cytosine (dCx) and/or 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine (dTx) in place of dC and dT residues in the EcoRII and MvaI recognition site CC(A/T)GG were synthesized in order to investigate specific recognition of the DNA sugar-phosphate backbone by EcoRII and MvaI restriction endonucleases. In 2'-deoxyxylosyl moieties of dCx and dTx, 3'-hydroxyl groups were inverted, which perturbs the related individual phosphates. Introduction of a single 2'-deoxyxylosyl moiety into a dC x dG pair resulted in a minor destabilization of double-stranded DNA structure. In the case of a dA x dT pair the effect of a 2'-deoxyxylose incorporation was much more pronounced. Multiple dCx modifications and their combination with dTx did not enhance the destabilization effect. Hydrolysis of dCx-containing DNA duplexes by EcoRII endonuclease was blocked and binding affinity was strongly depended on the location of an altered sugar. A DNA duplex containing a dTx residue was cleaved by the enzyme, but kcat/K(M) was slightly reduced. In contrast, MvaI endonuclease efficiently cleaved both types of sugar-altered substrate analogs. However it did not cleave conformationally perturbed scissile bonds, when the corresponding unmodified bonds were perfectly hydrolyzed in the same DNA duplexes. Based on these data the possible contributions of individual phosphates in the recognition site to substrate recognition and catalysis by EcoRII were proposed. We observed strikingly non-equivalent inputs for different phosphates with respect to their effect on EcoRII-DNA complex formation.     

Interaction of EcoRII restriction and modification enzyme with synthetic DNA fragments. IV. DNA duplexes with phosphoamide and pyrophosphate internucleotide bonds--the substrates for the study of single-strand breaks

A set of DNA duplexes with repeated EcoRII, EcoRI and AluI restriction endonuclease recognition sites in which EcoRII scissile phosphodiester bonds were replaced by phosphoramide or uncleavable pyrophosphate bonds have been synthesized. Endonuclease EcoRII was found not to cleave the substrate at the phosphoramide bond. The substrates containing non-nydrolysable pyrophosphate or phosphoramide bonds in one of the chains of EcoRII recognition sites were used to show that this enzyme is able to catalyze single-strand scissions. These scissions occur both in dA- and dT-containing chains of the recognition site. Endonuclease EcoRII interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of the other. Synthesized DNA-duplexes are cleaved specifically by EcoRI and AluI endonucleases, this cleavage being retarded if the modified bonds are in the recognition site (EcoRI) or flank it (AluI). For EcoRII and AluI this effect is more pronounced in the case of substrates with pyrophosphate bonds than with the phosphoramide ones.     

DNA-duplexes with phosphoamide bond: interaction with restriction endonucleases EcoRII and SsoII

We studied the interaction of EcoRII and SsoII restriction endonucleases with synthetic DNA duplexes, containing 3'N----5'P and 3'P----5'N phosphoamide internucleotide bonds in one of the cleavage points. Enzymatic hydrolysis of the modified strand of the duplexes is blocked in all cases. The presence of phosphoamide bonds was found to reduce the rate of cleavage of the natural strand by EcoRII and to have no influence in case of SsoII. Properties of the EcoRII endonuclease complex with its substrate, containing non-cleavable 3'N----5'P internucleotide bonds in each cleavage point, were examined. In the presence of Mg2+ ions the equilibrium association constant of the enzyme-substrate complex is 3-fold reduced, and the dissociation rate constant of the complex is increased by 1.5 times.     

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. X. Hydrolysis of substrates with structural abnormalities

Interaction of the EcoRII restriction endonuclease with a set of 30-membered substrates having structural anomalies in the recognition site (decreases CCT/AGG) and in adjacent sequences has been studied. A nick in the centre of the EcoRII recognition site between dC and dA residues slows down hydrolysis of the nonmodified strand, whereas the modified one is not cleaved. Removal of the phosphate group from the nick in this substrate does not alter the rate of the cleavage. The absence of one of the phosphate groups in the flanking sequence at a two-base-pair "distance" from the recognition site slows down the enzymatic hydrolysis. Removal of dA or dT out of the EcoRII recognition site blocks the enzymatic reaction. It appears that EcoRII does not interact with the phosphate group between dC and dA residues in the recognition site. Suggestions are made concerning possible contacts of the EcoRII restriction endonuclease with dA- and dT-residues of the recognition site and with the sugar-phosphate backbone of the adjacent nucleotide sequences.     

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.     

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.     

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.     

Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue in the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation.     

EcoRII can be activated to cleave refractory DNA recognition sites.

EcoRII restriction sites [5'-CC(A/T)GG] in phage T3 and T7 DNA are refractory to cleavage by EcoRII, but become sensitive to cleavage in the presence of DNAs which contain an abundance of EcoRII sensitive sites (e.g. pBR322 or lambda DNA). Studies using fragments of pBR322 containing different numbers of EcoRII sites show that the susceptibility to EcoRII cleavage is proportional to the number of sites in the individual fragment. We postulate that EcoRII is the prototype of restriction endonucleases which require at least 2 simultaneously bound substrate sites for their activation. EcoRII sites are refractory when they occur at relatively low frequency in the DNA. The restriction enzyme can be activated by DNA with a higher frequency of sites.