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.     

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.     

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.     

Iterative database searches demonstrate that glycoside hydrolase families 27, 31, 36 and 66 share a common evolutionary origin with family 13

A catalytic sequence motif PDX10-30(E/D)XK is found in many restriction enzymes. On the basis of sequence similarities and mapping of the conserved residues to the crystal structure of NgoMIV we suggest that residues D160, K182, R186, R188 and E195 contribute to the catalytic/DNA binding site of the Ecl18kI restriction endonuclease. Mutational analysis confirms the functional significance of the conserved residues of Ecl18kI. Therefore, we conclude that the active site motif 159VDX21KX12E of Ecl18kI differs from the canonical PDX10-30(E/D)XK motif characteristic for most of the restriction enzymes. Moreover, we propose that two subfamilies of endonucleases Ecl18kI/PspGI/EcoRII and Cfr10I/Bse634I/NgoMIV, specific, respectively, for CCNGG/CCWGG and RCCGGY/GCCGGC sites, share conserved active site architecture and DNA binding elements.     

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.     

DNA cleavage reactions by type II restriction enzymes that require two copies of their recognition sites

Several type II restriction endonucleases interact with two copies of their target sequence before they cleave DNA. Three such enzymes, NgoMIV, Cfr10I and NaeI, were tested on plasmids with one or two copies of their recognition sites, and on catenanes containing two interlinked rings of DNA with one site in each ring. The enzymes showed distinct patterns of behaviour. NgoMIV and NaeI cleaved the plasmid with two sites faster than that with one site and the catenanes at an intermediate rate, while Cfr10I gave similar steady-state rates on all three substrates. Both Cfr10I and NgoMIV converted the majority of the substrates with two sites directly to the products cut at both sites, while NaeI cleaved just one site at a time. All three enzymes thus synapse two DNA sites through three-dimensional space before cleaving DNA. With Cfr10I and NgoMIV, both sites are cleaved in one turnover, in a manner consistent with their tetrameric structures, while the cleavage of a single site by NaeI indicates that the second site acts not as a substrate but as an activator, as reported previously. The complexes spanning two sites have longer lifetimes on catenanes with one site in each ring than on circular DNA with two sites, which indicates that the catenanes have more freedom for site juxtaposition than plasmids with sites in cis.     

Specificity of new restrictases and methylases. Unusual modification of cytosine at position 4

Fourteen restriction endonucleases and 4 methylases were isolated and purified from 14 strains of Citrobacter freundii and Escherichia coli, which were isolated from natural sources. To determine the nucleotide sequence recognized by the endonucleases a comparison of DNA cleavage patterns, the evaluation of the cleavage frequency of some DNA with known recognition sequences and mapping was used. It was determined that Cfr101 is a new enzyme recognizing 5'PuCCGGPy. Other restriction enzymes isolated were isoschizomers of: Cfr5I, Cfr11I, Eco60I, Eco61I--EcoRII; Cfr4I, Cfr8I, Cfr13I--Sau96I; Cfr6I--PvuII, Cfr9I--SmaI, Eco26I--HgiJII; Eco32I--EcoRV; Eco52I--XmaIII; Eco56I--NaeI. Some of the enzymes in C. freundii and E. coli were found for the first time. The methylases MCfrI; MCfr6I, MCfr9I and MCfr10I recognize the same nucleotide sequence as specific endonucleases isolated from the same strain. DNA modification in vitro by MCfrI and MCfr10I yields 5-methylcytosine and 4-methylcytosine by MCfr6I and MCfr9I.     

Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family

The GIY-YIG nuclease domain was originally identified in homing endonucleases and enzymes involved in DNA repair and recombination. Many of the GIY-YIG family enzymes are functional as monomers. We show here that the Cfr42I restriction endonuclease which belongs to the GIY-YIG family and recognizes the symmetric sequence 5′-CCGC/GG-3′ (‘/’ indicates the cleavage site) is a tetramer in solution. Moreover, biochemical and kinetic studies provided here demonstrate that the Cfr42I tetramer is catalytically active only upon simultaneous binding of two copies of its recognition sequence. In that respect Cfr42I resembles the homotetrameric Type IIF restriction enzymes that belong to the distinct PD-(E/D)XK nuclease superfamily. Unlike the PD-(E/D)XK enzymes, the GIY-YIG nuclease Cfr42I accommodates an extremely wide selection of metal-ion cofactors, including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺, Cu²⁺ and Ca²⁺. To our knowledge, Cfr42I is the first tetrameric GIY-YIG family enzyme. Similar structural arrangement and phenotypes displayed by restriction enzymes of the PD-(E/D)XK and GIY-YIG nuclease families point to the functional significance of tetramerization.     

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

Restriction endonucleases of the PD…D/EXK family need Mg²⁺ for DNA cleavage. Whereas Mg²⁺ (or Mn²⁺) promotes catalysis, Ca²⁺ (without Mg²⁺) only supports DNA binding. The role of Mg²⁺ in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg²⁺ involved in catalysis. To address this problem, we measured the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me²⁺ per active site. DNA cleavage experiments were carried out at various Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg²⁺ and Mn²⁺ concentration dependence. In general, the Mg²⁺ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn²⁺ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg²⁺ or Mn²⁺ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca²⁺. Based on these results, we propose that one Mg²⁺ or Mn²⁺ is critical for restriction enzyme activation, and binding of a second Me²⁺ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg²⁺ or Mn²⁺ mainly leads to an increase in K_m, such that the inhibitory effect of excess Mg²⁺ or Mn²⁺ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me²⁺ binding to these enzymes.     

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.     

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.     

The Cfr10I restriction enzyme is functional as a tetramer.

It is thought that most of the type II restriction endonucleases interact with DNA as homodimers. Cfr10I is a typical type II restriction enzyme that recognises the 5'-Pu decreases CCGGPy sequence and cleaves it as indicated by the arrow. Gel-filtration and analytical ultracentrifugation data presented here indicate that Cfr10I is a homotetramer in isolation. The only SfiI restriction enzyme that recognises the long interrupted recognition sequence 5'-GGCCNNNNNGGCC has been previously reported to operate as a tetramer however, its structure is unknown. Analysis of Cfr10I crystals revealed that a single molecule in the asymmetric unit is repeated by D2 symmetry to form a tetramer. To determine whether the packing of the Cfr10I in the crystal reflects the quaternary structure of the protein in solution, the tryptophan W220 residue located at the putative dimer-dimer interface was mutated to alanine, and the structural and functional consequences of the substitution were analysed. Equilibrium sedimentation experiments revealed that, in contrast to the wild-type Cfr10I, the W220A mutant exists in solution predominantly as a dimer. In addition, the tetramer seems to be a catalytically important form of Cfr10I, since the DNA cleavage activity of the W220A mutant is < 0.1% of that of the wild-type enzyme. Further, analysis of plasmid DNA cleavage suggests that the Cfr10I tetramer is able to interact with two copies of the recognition sequence, located on the same DNA molecule. Indeed, electron microscopy studies demonstrated that two distant recognition sites are brought together through the DNA looping induced by the simultaneous binding of the Cfr10I tetramer to both sites. These data are consistent with the tetramer being a functionally important form of Cfr10I.     

Cleavage of synthetic substrates containing non-nucleotide inserts by restriction endonucleases. Change in the cleavage specificity of endonuclease SsoII.

A study was made of the interaction between restriction endonucleases recognizing CCNGG (SsoII and ScrFI) or CCA/TGG (MvaI and EcoRII) DNA sequences and a set of synthetic substrates containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy] methylguanine (gIG) residues replacing either one of the central nucleosides or dG residues in the recognition site. The non-nucleotide inserts (except for gIG) introduced into the recognition site both increase the efficiency of SsoII and change its specificity. A cleavage at the noncanonical position takes place, in some cases in addition to the correct ones. Noncanonical hydrolysis by SsoII occurs at the phosphodiester bond adjacent to the point of modification towards the 5'-end. With the guanine base returned (the substrate with gIG), the correct cleavage position is restored. ScrFI specifically cleaves all the modified substrates. DNA duplexes with non-nucleotide inserts (except for the gIG-containing duplex) are resistant to hydrolysis by MvaI and EcoRII. Prompted by the data obtained we discuss the peculiarities of recognition by restriction endonucleases of 5-membered DNA sequences which have completely or partially degenerated central base pairs. It is suggested that SsoII forms a complex with DNA in an 'open' form.     

Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence

Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI–DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution.     

Analysis of DNA looping interactions by type II restriction enzymes that require two copies of their recognition sites

Before cleaving DNA substrates with two recognition sites, the Cfr10I, NgoMIV, NaeI and SfiI restriction endonucleases bridge the two sites through 3D space, looping out the intervening DNA. To characterise their looping interactions, the enzymes were added to plasmids with two recognition sites interspersed with two res sites for site-specific recombination by Tn21 resolvase, in buffers that contained either EDTA or CaCl2 so as to preclude DNA cleavage by the endonuclease; the extent to which the res sites were sequestered into separate loops was evaluated from the degree of inhibition of resolvase. With Cfr10I, a looped complex was detected in the presence but not in the absence of Ca(2+); it had a lifetime of about 90 seconds. Neither NgoMIV nor NaeI gave looped complexes of sufficient stability to be detected by this method. In contrast, SfiI with Ca(2+) produced a looped complex that survived for more than seven hours, whereas its looping interaction in EDTA lasts for about four minutes. When resolvase was added to a SfiI binding reaction in EDTA followed immediately by CaCl2, the looped DNA was blocked from recombination while the unlooped DNA underwent recombination. By measuring the distribution between looped and unlooped DNA at various SfiI concentrations, and by fitting the data to a model for DNA binding by a tetrameric protein to two sites in cis, an equilibrium constant for the looping interaction was determined. The equilibrium constant was essentially independent of the length of DNA between the SfiI sites.     

Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease

Restricion endonuclease Ecl18kI is specific for the sequence /CCNGG and cleaves it before the outer C to generate 5 nt 5'-overhangs. It has been suggested that Ecl18kI is evolutionarily related to NgoMIV, a 6-bp cutter that cleaves the sequence G/CCGGC and leaves 4 nt 5'-overhangs. Here, we report the crystal structure of the Ecl18kI-DNA complex at 1.7 A resolution and compare it with the known structure of the NgoMIV-DNA complex. We find that Ecl18kI flips both central nucleotides within the CCNGG sequence and buries the extruded bases in pockets within the protein. Nucleotide flipping disrupts Watson-Crick base pairing, induces a kink in the DNA and shifts the DNA register by 1 bp, making the distances between scissile phosphates in the Ecl18kI and NgoMIV cocrystal structures nearly identical. Therefore, the two enzymes can use a conserved DNA recognition module, yet recognize different sequences, and form superimposable dimers, yet generate different cleavage patterns. Hence, Ecl18kI is the first example of a restriction endonuclease that flips nucleotides to achieve specificity for its recognition site.     

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.     

The structure of SgrAI bound to DNA; recognition of an 8 base pair target

The three-dimensional X-ray crystal structure of the ‘rare cutting’ type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca²⁺ bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn²⁺, with strong binding at the protein–DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact to the G from an arginine side chain located in a region unique to SgrAI.     

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.