Subunit assembly for DNA cleavage by restriction endonuclease SgrAI

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

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.     

Communications between catalytic sites in the protein-DNA synapse by the SfiI endonuclease

The SfiI endonuclease is a tetrameric protein with two DNA-binding clefts. It has to bind two copies of its recognition sequence, one at each cleft, before it cleaves DNA. While SfiI binds cooperatively to two cognate sites, it binds only one non-cognate DNA molecule at a time and the resultant complex is precluded from binding cognate DNA at the vacant cleft. To examine the communications between separate binding sites in a protein that synapses two segments of DNA, SfiI was tested with oligonucleotide duplexes containing its recognition sequence but with either R(p) or S(p) phosphorothioate linkages at the scissile bonds. Though SfiI has low activity on the R(p) and none against the S(p) diastereoisomer, it bound these duplexes in the same cooperative manner as oxyester duplexes, though with a reduced affinity for the S(p) derivative. It also formed complexes with one phosphorothioate-duplex and one oxyester-duplex but, when Mg(2+) was added to the hybrid complexes, the phosphorothioate moiety at one DNA-binding cleft prevented the enzyme from cleaving the oxyester duplex at the other cleft. SfiI is thus restrained from catalytic action until it recognises the correct nucleotide sequence at two DNA loci and the correct phosphodiester functions at both loci.     

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.     

Domain swapping in allosteric modulation of DNA specificity

SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of cleavage activity and sequence specificity. Previous studies have shown that DNA bound dimers of SgrAI oligomerize into an activated form with higher DNA cleavage rates, although previously determined crystal structures of SgrAI bound to DNA show only the DNA bound dimer. A new crystal structure of the type II restriction endonuclease SgrAI bound to DNA and Ca(2+) is now presented, which shows the close association of two DNA bound SgrAI dimers. This tetrameric form is unlike those of the homologous enzymes Cfr10I and NgoMIV and is formed by the swapping of the amino-terminal 24 amino acid residues. Two mutations predicted to destabilize the swapped form of SgrAI, P27W and P27G, have been made and shown to eliminate both the oligomerization of the DNA bound SgrAI dimers as well as the allosteric stimulation of DNA cleavage by SgrAI. A mechanism involving domain swapping is proposed to explain the unusual allosteric properties of SgrAI via association of the domain swapped tetramer of SgrAI bound to DNA into higher order oligomers.     

Specificity from the synapsis of DNA elements by the Sfi I endonuclease.

The synapsis of DNA sites is a prerequisite for the reactions of many proteins that act at specific DNA sequences. The requirement for synapsis was investigated by analysing the reactions of Sfi I, a tetrameric restriction enzyme that cleaves DNA only after interacting with two recognition sites. In the presence of Mg2+, oligonucleotide duplexes with the cognate recognition sequence were cleaved rapidly, with cooperative kinetics, while non-cognate duplexes were not cleaved. In the absence of Mg2+, the primary complex formed by Sfi I with cognate DNA contained two duplexes synapsed by the tetramer: a secondary complex containing one duplex was seen only at elevated Sfi I concentrations. In contrast, the principal complex with non-cognate DNA contained one duplex bound to Sfi I. Pairs of non-cognate duplexes, or one cognate and one non-cognate duplex, generally failed to form synaptic complexes. On adding Mg2+to complexes with cognate DNA, cleavage occurred much more rapidly in the synaptic complex than in the secondary complex. DNA synapsis thus acts to enhance the specificity of Sfi I for its recognition sequence, by demanding two cognate sites for a catalytically active complex and by excluding non-cognate sites from the synaptic complex.     

Probing the Run-On Oligomer of Activated SgrAI Bound to DNA

SgrAI is a type II restriction endonuclease with an unusual mechanism of activation involving run-on oligomerization. The run-on oligomer is formed from complexes of SgrAI bound to DNA containing its 8 bp primary recognition sequence (uncleaved or cleaved), and also binds (and thereby activates for DNA cleavage) complexes of SgrAI bound to secondary site DNA sequences which contain a single base substitution in either the 1^st/8^th or the 2^nd/7^th position of the primary recognition sequence. This modulation of enzyme activity via run-on oligomerization is a newly appreciated phenomenon that has been shown for a small but increasing number of enzymes. One outstanding question regarding the mechanistic model for SgrAI is whether or not the activating primary site DNA must be cleaved by SgrAI prior to inducing activation. Herein we show that an uncleavable primary site DNA containing a 3’-S-phosphorothiolate is in fact able to induce activation. In addition, we now show that cleavage of secondary site DNA can be activated to nearly the same degree as primary, provided a sufficient number of flanking base pairs are present. We also show differences in activation and cleavage of the two types of secondary site, and that effects of selected single site substitutions in SgrAI, as well as measured collisional cross-sections from previous work, are consistent with the cryo-electron microscopy model for the run-on activated oligomer of SgrAI bound to DNA.     

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.     

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.     

DNA nicking by HinP1I endonuclease: bending, base flipping and minor groove expansion

HinP1I recognizes and cleaves the palindromic tetranucleotide sequence G↓CGC in DNA. We report three structures of HinP1I–DNA complexes: in the presence of Ca²⁺ (pre-reactive complex), in the absence of metal ion (binary complex) and in the presence of Mg²⁺ (post-reactive complex). HinP1I forms a back-to-back dimer with two active sites and two DNA duplexes bound on the outer surfaces of the dimer facing away from each other. The 10 bp DNA duplexes undergo protein-induced distortions exhibiting features of A-, B- and Z-conformations: bending on one side (by intercalation of a phenylalanine side chain into the major groove), base flipping on the other side of the recognition site (by expanding the step rise distance of the local base pair to Z-form) and a local A-form conformation between the two central C:G base pairs of the recognition site (by binding of the N-terminal helix in the minor groove). In the pre- and post-reactive complexes, two metals (Ca²⁺ or Mg²⁺) are found in the active site. The enzyme appears to cleave DNA sequentially, hydrolyzing first one DNA strand, as seen in the post-reactive complex in the crystalline state, and then the other, as supported by the observation that, in solution, a nicked DNA intermediate accumulates before linearization.     

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

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

Kinetic analysis of the coordinated interaction of SgrAI restriction endonuclease with different DNA targets

SgrAI restriction endonuclease cooperatively interacts and cleaves two target sites that include both the canonical sites, CPuCCGGPyG, and the secondary sites, CPuCCGGPy(A/T/C). It has been observed that the cleaved canonical sites stimulate SgrAI cleavage at the secondary sites. Equilibrium binding studies show that SgrAI binds to its canonical sites with a high affinity (Ka = 4-8 x 10(10) M-1) and that it has a 15-fold lower affinity for the cleaved canonical sites and a 30-fold lower affinity for the secondary sites. Steady-state kinetics reveals substrate cooperativity for SgrAI cleavage on both canonical and secondary sites. The specificity of SgrAI for the secondary site CACCGGCT, as measured by kcat/K is about 500-fold lower than that for the canonical site CACCGGCG, but this difference is reduced to 10-fold in the presence of the cleaved canonical sites. The efficiency of canonical site cleavage also increases by 3-fold when the cleaved canonical sites are present in the reaction. Furthermore, the substrate cooperativity for SgrAI cleavage is abolished for both types of sites in the presence of cleaved canonical sites. These results indicate that target site cleavage occurs via a coordinated interaction of two SgrAI protein subunits, where the subunit bound to the cleaved site stimulates the cleavage of the uncut site bound by the other subunit. The free subunits of SgrAI have the flexibility to bind different target sites and, consequently, assemble into various catalytically active complexes, which differ in their catalytic efficiencies.     

Crystallization of complexes of EcoRV endonuclease with cognate and non-cognate DNA fragments

Complexes of the type II restriction endonuclease EcoRV with a variety of short, selfcomplementary deoxyoligonucleotides have been crystallized. The best crystals diffract to about 2.7 A resolution and consist of 1:1 complexes between endonuclease dimers and duplexes of the cognate decamer GGGATATCCC containing the hexameric RV recognition sequence GATATC. Crystals with the non-cognate DNA octamer duplexes CGAGCTCG and CGAATTCG diffract to 3.0 and 3.5 A resolution, respectively, and contain two DNA duplexes per enzyme dimer.     

Fidelity of DNA sequence recognition by the SfiI restriction endonuclease is determined by communications between its two DNA-binding sites

The SfiI restriction endonuclease is a tetramer in which two subunits form a dimeric unit that contains one DNA binding cleft and the other two subunits contain a second cleft on the opposite side of the protein. Full activity requires both clefts to be filled with its recognition sequence: SfiI has low activity when bound to one site. The ability of SfiI to cleave non-cognate sites, one base pair different from the true site, was initially tested on substrates that lacked specific sites but which contained either one or multiple non-cognate sites. No cleavage of the DNA with one non-cognate site was detected, while a small fraction of the DNA with multiple sites was nicked. The alternative sequences were, however, cleaved in both strands, albeit at low levels, when the DNA also carried either a recognition site for SfiI or the termini generated by SfiI. Further tests employed a mutant of SfiI, altered at the dimer interface, which was known to be more active than wild-type SfiI when bound to a single site. This mutant similarly failed to cleave DNA with one non-cognate site, but cleaved the substrates with multiple non-cognate sites more readily than did the native enzyme. To cleave additional sites, SfiI thus needs to interact concurrently with either two non-cognate sites or one non-cognate and one cognate site (or the termini thereof), yet this arrangement is still restrained from cleaving the alternative site unless the communication pathway between the two DNA-binding clefts is disrupted.     

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+.     

Conversion of the tetrameric restriction endonuclease Bse634I into a dimer: oligomeric structure-stability-function correlations

The Bse634I restriction endonuclease is a tetramer and belongs to the type IIF subtype of restriction enzymes. It requires two recognition sites for its optimal activity and cleaves plasmid DNA with two sites much faster than a single-site DNA. We show that disruption of the tetramerisation interface of Bse634I by site-directed mutagenesis converts the tetrameric enzyme into a dimer. Dimeric W228A mutant cleaves plasmid DNA containing one or two sites with the same efficiency as the tetramer cleaves the two-site plasmid. Hence, the catalytic activity of the Bse634I tetramer on a single-site DNA is down-regulated due to the cross-talking interactions between the individual dimers. The autoinhibition within the Bse634I tetramer is relieved by bridging two DNA copies into the synaptic complex that promotes fast and concerted cleavage at both sites. Cleavage analysis of the oligonucleotide attached to the solid support revealed that Bse634I is able to form catalytically competent synaptic complexes by bridging two molecules of the cognate DNA, cognate DNA-miscognate DNA and cognate DNA-product DNA. Taken together, our data demonstrate that a single W228A mutation converts a tetrameric type IIF restriction enzyme Bse634I into the orthodox dimeric type IIP restriction endonuclease. However, the stability of the dimer towards chemical denaturants, thermal inactivation and proteolytic degradation are compromised.     

Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease

The BcgI endonuclease exemplifies a subset of restriction enzymes, the Type IIB class, which make two double-strand breaks (DSBs) at each copy of their recognition sequence, one either side of the site, to excise the sequence from the remainder of the DNA. In this study, we show that BcgI is essentially inactive when bound to a single site and that to cleave a DNA with one copy of its recognition sequence, it has to act in trans, bridging two separate DNA molecules. We also show that BcgI makes the two DSBs at an individual site in a highly concerted manner. Intermediates cut on one side of the site do not accumulate during the course of the reaction: instead, the DNA is converted straight to the final products cut on both sides. On DNA with two sites, BcgI bridges the sites in cis and then generally proceeds to cut both strands on both sides of both sites without leaving the DNA. The BcgI restriction enzyme can thus excise two DNA segments together, by cleaving eight phosphodiester bonds within a single-DNA binding event.     

Protein assembly and DNA looping by the FokI restriction endonuclease

The FokI restriction endonuclease recognizes an asymmetric DNA sequence and cuts both strands at fixed positions upstream of the site. The sequence is contacted by a single monomer of the protein, but the monomer has only one catalytic centre and forms a dimer to cut both strands. FokI is also known to cleave DNA with two copies of its site more rapidly than DNA with one copy. To discover how FokI acts at a single site and how it acts at two sites, its reactions were examined on a series of plasmids with either one recognition site or with two sites separated by varied distances, sometimes in the presence of a DNA-binding defective mutant of FokI. These experiments showed that, to cleave DNA with one site, the monomer bound to that site associates via a weak protein–protein interaction with a second monomer that remains detached from the recognition sequence. Nevertheless, the second monomer catalyses phosphodiester bond hydrolysis at the same rate as the DNA-bound monomer. On DNA with two sites, two monomers of FokI interact strongly, as a result of being tethered to the same molecule of DNA, and sequester the intervening DNA in a loop.     

Restriction endonucleases that bridge and excise two recognition sites from DNA

Most restriction endonucleases bridge two target sites before cleaving DNA: examples include all of the translocating Type I and Type III systems, and many Type II nucleases acting at their sites. A subset of Type II enzymes, the IIB systems, recognise bipartite sequences, like Type I sites, but cut specified phosphodiester bonds near their sites, like Type IIS enzymes. However, they make two double-strand breaks, one either side of the site, to release the recognition sequence on a short DNA fragment; 34 bp long in the case of the archetype, BcgI. It has been suggested that BcgI needs to interact with two recognition sites to cleave DNA but whether this is a general requirement for Type IIB enzymes had yet to be established. Ten Type IIB nucleases were tested against DNA substrates with one or two copies of the requisite sequences. With one exception, they all bridged two sites before cutting the DNA, usually in concerted reactions at both sites. The sites were ideally positioned in cis rather than in trans and were bridged through 3-D space, like Type II enzymes, rather than along the 1-D contour of the DNA, as seen with Type I enzymes. The standard mode of action for the restriction enzymes that excise their recognition sites from DNA thus involves concurrent action at two DNA sites.