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.     

Recombination by resolvase to analyse DNA communications by the SfiI restriction endonuclease.

The SfiI endonuclease differs from other type II restriction enzymes by cleaving DNA concertedly at two copies of its recognition site, its optimal activity being with two sites on the same DNA molecule. The nature of this communication event between distant DNA sites was analysed on plasmids with recognition sites for SfiI interspersed with recombination sites for resolvase. These were converted by resolvase to catenanes carrying one SfiI site on each ring. The catenanes were cleaved by SfiI almost as readily as a single ring with two sites, in contrast to the slow reactions on DNA rings with one SfiI site. Interactions between SfiI sites on the same DNA therefore cannot follow the DNA contour and, instead, must stem from their physical proximity. In buffer lacking Mg2+, where SfiI is inactive while resolvase is active, the addition of SfiI to a plasmid with target sites for both proteins blocked recombination by resolvase, due to the restriction enzyme bridging its sites and thus isolating the sites for resolvase into separate loops. The extent of DNA looping by SfiI matched its extent of DNA cleavage in the presence of Mg2+.     

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.     

Repercussions of DNA tracking by the type IC restriction endonuclease EcoR124I on linear, circular and catenated substrates.

Type I restriction endonucleases such as EcoR124I cleave DNA at undefined loci, distant from their recognition sequences, by a mechanism that involves the enzyme tracking along the DNA between recognition and cleavage sites. This mechanism was examined on plasmids that carried recognition sites for EcoR124I and recombination sites for resolvase, the latter to create DNA catenanes. Supercoiled substrates with either one or two restriction sites were linearized by EcoR124I at similar rates, although the two-site molecule underwent further cleavage more readily than the one-site DNA. The catenane from the plasmid with one EcoR124I site, carrying the site on the smaller of the two rings, was cleaved by EcoR124I exclusively in the small ring, and this underwent multiple cleavage akin to the two-site plasmid. Linear substrates derived from the plasmids were cleaved by EcoR124I at very slow rates. The communication between recognition and cleavage sites therefore cannot stem from random looping. Instead, it must follow the DNA contour between the sites. On a circular DNA, the translocation of non-specific DNA past the specifically bound protein should increase negative supercoiling in one domain and decrease it in the other. The ensuing topological barrier may be the trigger for DNA cleavage.     

DNA communications by Type III restriction endonucleases--confirmation of 1D translocation over 3D looping

DNA cleavage by Type III restriction enzymes is governed strictly by the relative arrangement of recognition sites on a DNA substrate—endonuclease activity is usually only triggered by sequences in head-to-head orientation. Tens to thousands of base pairs can separate these sites. Long distance communication over such distances could occur by either one-dimensional (1D) DNA translocation or 3D DNA looping. To distinguish between these alternatives, we analysed the activity of EcoPI and EcoP15I on DNA catenanes in which the recognition sites were either on the same or separate rings. While substrates with a pair of sites located on the same ring were cleaved efficiently, catenanes with sites on separate rings were not cleaved. These results exclude a simple 3D DNA-looping activity. To characterize the interactions further, EcoPI was incubated with plasmids carrying two recognition sites interspersed with two 21res sites for site-specific recombination by Tn21 resolvase; inhibition of recombination would indicate the formation of stable DNA loops. No inhibition was observed, even under conditions where EcoPI translocation could also occur.     

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.     

Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA.

Type IIs restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions, typically several base pairs away from the recognition site. These enzymes are generally monomers that transiently associate to form dimers to cleave both strands. Their reactions could involve bridging interactions between two copies of their recognition sequence. To examine this possibility, several type IIs enzymes were tested against substrates with either one or two target sites. Some of the enzymes cleaved the DNA with two target sites at the same rate as that with one site, but most cut their two-site substrate more rapidly than the one-site DNA. In some cases, the two sites were cut sequentially, at rates that were equal to each other but that exceeded the rate on the one-site DNA. In another case, the DNA with two sites was cleaved rapidly at one site, but the residual site was cleaved at a much slower rate. In a further example, the two sites were cleaved concertedly to give directly the final products cut at both sites. Many type IIs enzymes thus interact with two copies of their recognition sequence before cleaving DNA, although via several different mechanisms.     

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.     

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.     

Site-specific relaxation and recombination by the Tn3 resolvase: recognition of the DNA path between oriented res sites

We studied the dynamics of site-specific recombination by the resolvase encoded by the Escherichia coli transposon Tn3. The pure enzyme recombined supercoiled plasmids containing two directly repeated recombination sites, called res sites. Resolvase is the first strictly site-specific topoisomerase. It relaxed only plasmids containing directly repeated res sites; substrates with zero, one or two inverted sites were inert. Even when the proximity of res sites was ensured by catenation of plasmids with a single site, neither relaxation nor recombination occurred. The two circular products of recombination were catenanes interlinked only once. These properties of resolvase require that the path of the DNA between res sites be clearly defined and that strand exchange occur with a unique geometry. A model in which one subunit of a dimeric resolvase is bound at one res site, while the other searches along adjacent DNA until it encounters the second site, would account for the ability of resolvase to distinguish intramolecular from intermolecular sites, to sense the relative orientation of sites and to produce singly interlinked catenanes. Because resolvase is a type 1 topoisomerase, we infer that it makes the required duplex bDNA breaks of recombination one strand at a time.     

Long-range communications between DNA sites by the dimeric restriction endonuclease SgrAI

The SgrAI endonuclease displays its maximal activity on DNA with two copies of its recognition sequence, cleaving both sites concertedly. While most restriction enzymes that act concurrently at two sites are tetramers, SgrAI is a dimer in solution. Its reaction at two cognate sites involves the association of two DNA-bound dimers. SgrAI can also bridge cognate and secondary sites, the latter being certain sequences that differ from the cognate by one base-pair. The mechanisms for cognate-cognate and cognate-secondary communications were examined for sites in the following topological relationships: in cis, on plasmids with two sites in a single DNA molecule; on catenanes containing two interlinked rings of DNA with one site in each ring; and in trans, on oligoduplexes carrying either a single site or the DNA termini generated by SgrAI. Both cognate-cognate and cognate-secondary interactions occur through 3-D space and not by 1-D tracking along the DNA. Both sorts of communication arise more readily when the sites are tethered to each other, either in cis on the same molecule of DNA or by the interlinking of catenane rings, than when released from the tether. However, the dimer bound to an oligoduplex carrying either a cognate or a secondary site could be activated to cleave that duplex by interacting with a second dimer bound to the recognition site, provided both duplexes are at least 30 base-pairs long: the second dimer could alternatively be bound to the two duplexes that correspond to the products of DNA cleavage by SgrAI.     

Reactions of type II restriction endonucleases with 8-base pair recognition sites

Type II restriction endonucleases usually recognize 4-6-base pair (bp) sites on DNA and cleave each site in a separate reaction. A few type II endonucleases have 8-bp recognition sites, but these seem unsuited for restriction, since their sites are rare on most DNA. Moreover, only one endonuclease that recognizes a target containing 8 bp has been examined to date, and this enzyme, SfiI, needs two copies of this site for its DNA cleavage reaction. In this study, several endonucleases with 8-bp sites were tested on plasmids that have either one or two copies of the relevant sequence to determine if they also need two sites. SgfI, SrfI, FseI, PacI, PmeI, Sse8781I, and SdaI all acted through equal and independent reactions at each site. AscI cleaved the DNA with one site at the same rate as that with two sites but acted processively on the latter. In contrast, SgrAI showed a marked preference for the plasmid with two sites and cleaved both sites on this DNA in a concerted manner, like SfiI. Endonucleases that require two copies of an 8-bp sequence may be widespread in nature, where, despite this seemingly inappropriate requirement, they may function in DNA restriction.     

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 type IIs restriction endonuclease BspMI is a tetramer that acts concertedly at two copies of an asymmetric DNA sequence.

Type IIs endonucleases recognize asymmetric DNA sequences and cleave both strands at fixed positions downstream of the sequence. Many type IIs enzymes, including BspMI, cleave substrates with two sites more rapidly than those with one site. They usually act sequentially on DNA with two sites, but BspMI converted such a substrate directly to the final products cut at both sites. The BspMI endonuclease was found to be a tetramer, in contrast to the monomeric structures for many type IIs enzymes. No change in subunit association occurred during the BspMI reaction. Plasmids with two BspMI sites were cleaved in cis, in reactions spanning sites in the same DNA, even when the sites were separated by just 38 bp. Plasmids with one BspMI site were cleaved in trans, with the enzyme bridging sites in separate DNA molecules: these slow reactions could be accelerated by adding a second DNA with the recognition sequence. Thus, whereas many type IIs enzymes dimerize before cleaving DNA, a process facilitated by two recognition sites in cis, the BspMI tetramer binds two copies of its recognition sequence before cleaving the DNA in both strands at both sites.     

One recognition sequence, seven restriction enzymes, five reaction mechanisms

The diversity of reaction mechanisms employed by Type II restriction enzymes was investigated by analysing the reactions of seven endonucleases at the same DNA sequence. NarI, KasI, Mly113I, SfoI, EgeI, EheI and BbeI cleave DNA at several different positions in the sequence 5′-GGCGCC-3′. Their reactions on plasmids with one or two copies of this sequence revealed five distinct mechanisms. These differ in terms of the number of sites the enzyme binds, and the number of phosphodiester bonds cleaved per turnover. NarI binds two sites, but cleaves only one bond per DNA-binding event. KasI also cuts only one bond per turnover but acts at individual sites, preferring intact to nicked sites. Mly113I cuts both strands of its recognition sites, but shows full activity only when bound to two sites, which are then cleaved concertedly. SfoI, EgeI and EheI cut both strands at individual sites, in the manner historically considered as normal for Type II enzymes. Finally, BbeI displays an absolute requirement for two sites in close physical proximity, which are cleaved concertedly. The range of reaction mechanisms for restriction enzymes is thus larger than commonly imagined, as is the number of enzymes needing two recognition sites.     

Modified DNA fragments activate NaeI cleavage of refractory DNA sites.

Endonuclease NaeI cleaves DNA using a two-site mechanism. The DNA-binding sites are nonidentical: they recognize different families of flanking sequences. A unique NaeI site that is resistant to cleavage resides in M13 double-stranded DNA. NaeI can be activated to cleave this site by small DNA fragments containing one or more NaeI sites. These activators are not practical for genetic engineering because unphosphorylated activators that are consumed during the cleavage of substrate give ends that may interfere with subsequent ligations. We show that a DNA fragment containing phosphorothioate linkages at the NaeI scissile bonds (S-activator) is not cleaved by NaeI, even though this S-activator binds to the substrate site. The S-activator activates NaeI to cleave M13 DNA under conditions that completely exhaust unsubstituted activator. These results demonstrate that activation is not coupled to cleavage of activator, that NaeI reverts to its inactive state soon after dissociation of the EA complex, and that S-activator makes for a nondepletable activator during prolonged incubations.     

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.     

Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA

The crystal structure of the NgoMIV restriction endonuclease in complex with cleaved DNA has been determined at 1.6 A resolution. The crystallographic asymmetric unit contains a protein tetramer and two DNA molecules cleaved at their recognition sites. This is the first structure of a tetrameric restriction enzyme-DNA complex. In the tetramer, two primary dimers are arranged back to back with two oligonucleotides bound in clefts on opposite sides of the tetramer. The DNA molecules retain a B-type conformation and have an enclosed angle between their helical axes of 60 degrees. Sequence-specific interactions occur in both the major and minor grooves. Two Mg2+ ions are located close to the cleaved phosphate at the active site of NgoMIV. Biochemical experiments show that interactions between the recognition sites within the tetramer greatly increase DNA cleavage efficiency.     

The metal-independent type IIs restriction enzyme BfiI is a dimer that binds two DNA sites but has only one catalytic centre

BfiI is a novel type IIs restriction endonuclease that, unlike all other restriction enzymes characterised to date, cleaves DNA in the absence of Mg(2+). The amino acid sequence of the N-terminal part of BfiI has some similarities to Nuc of Salmonella typhimurium, an EDTA-resistant nuclease akin to phospholipase D. The dimeric form of Nuc contains a single active site composed of residues from both subunits. To examine the roles of the amino acid residues of BfiI that align with the catalytic residues in Nuc, a set of alanine replacement mutants was generated by site-directed mutagenesis. The mutationally altered forms of BfiI were all catalytically inactive but were still able to bind DNA specifically. The active site of BfiI is thus likely to be similar to that of Nuc. BfiI was also found by gel-filtration to be a dimer in solution. Both gel-shift and pull-down assays indicated that the dimeric form of BfiI binds two copies of its recognition sequence. In reactions on plasmids with either one or two copies of its recognition sequence, BfiI cleaved the DNA with two sites more rapidly than that with one site. Yet, when bound to two copies of its recognition sequence, the BfiI dimer cleaved only one phosphodiester bond at a time. The dimer thus seems to contain two DNA-binding domains but only one active site.     

Phage P1 Cre-loxP site-specific recombination. Effects of DNA supercoiling on catenation and knotting of recombinant products

Bacteriophage P1 contains a site-specific recombination system consisting of a site, loxP, and a recombinase protein Cre. We have shown that with purified Cre protein we can carry out recombination between two loxP sites in vitro. When that recombination occurs between two sites in direct orientation on the same DNA molecule, we observed the production of free and catenated circular molecules. In this paper we show that recombination between sites in opposite orientation leads to both knotted and unknotted circular products. We also demonstrate that the production of catenanes and knots is influenced by two factors: (1) supercoiling in the DNA substrate, supercoiled DNA substrates yield significantly more catenated and knotted products than nicked circular substrates; and (2) mutations in the loxP site, a class of mutations have been isolated that carry out recombination but result in a distribution of products in which the ratio of catenanes to free circles is increased over that observed with a wild-type site. A more detailed analysis of the products from recombination between wild-type sites indicates: (1) that the catenanes or knots produced by recombination are both simple and complex; (2) that the ratio of free products to catenanes is independent of the distance between the two directly repeated loxP sites; and (3) that for DNA substrates with four loxP sites significant recombination between non-adjacent sites occurs to give free circular products. These observations provide insights into how two loxP sites are brought together during recombination.