The energetics of the interaction of BamHI endonuclease with its recognition site GGATCC.

The interaction of BamHI endonuclease with DNA has been studied crystallographically, but has not been characterized rigorously in solution. The enzyme binds in solution as a homodimer to its recognition site GGATCC. Only six base-pairs are directly recognized, but binding affinity (in the absence of the catalytic cofactor Mg(2+)) increases 5400-fold as oligonucleotide length increases from 10 to 14 bp. Binding is modulated by sequence context outside the recognition site, varying about 30-fold from the bes t (GTG or TAT) to the worst (CGG) flanking triplets. BamHI, EcoRI and EcoRV endonucleases all have different context preferences, suggesting that context affects binding by influencing the free energy levels of the complexes rather than that of the free DNA. Ethylation interference footprinting in the absence of divalent metal shows a localized and symmetrical pattern of phosphate contacts, with strong contacts at NpNpNpGGApTCC. In the presence of Mg(2+), first-order cleavage rate constants are identical in the two GGA half-sites, are the same for the two nicked intermediates and are unaffected by substrate length in the range 10-24 bp. DNA binding is strongly enhanced by mutations D94N, E111A or E113K, by binding of Ca(2+) at the active site, or by deletion of the scissile phosphate GpGATCC, indicating that a cluster of negative charges at the catalytic site contributes at least 3-4 kcal/mol of unfavorable binding free energy. This electrostatic repulsion destabilizes the enzyme-DNA complex and favors metal ion binding and progression to the transition state for cleavage.

Structure of free BglII reveals an unprecedented scissor-like motion for opening an endonuclease.

Restriction endonuclease BglII completely encircles its target DNA, making contacts to both the major and minor grooves. To allow the DNA to enter and leave the binding cleft, the enzyme dimer has to rearrange. To understand how this occurs, we have solved the structure of the free enzyme at 2.3 A resolution, as a complement to our earlier work on the BglII-DNA complex. Unexpectedly, the enzyme opens by a dramatic 'scissor-like' motion, accompanied by a complete rearrangement of the alpha-helices at the dimer interface. Moreover, within each monomer, a set of residues--a 'lever'--lowers or raises to alternately sequester or expose the active site residues. Such an extreme difference in free versus complexed structures has not been reported for other restriction endonucleases. This elegant mechanism for capturing DNA may extend to other enzymes that encircle DNA.

Crystallization and preliminary X-ray diffraction analysis of MspI restriction endonuclease in complex with its cognate DNA.

The MspI restriction endonuclease is a type II restriction enzyme. Unlike all other restriction enzymes with known structures, MspI recognizes the palindromic tetranucleotide sequence 5'-C/CGG and cleaves it as indicated by the '/' to produce DNA products with 5' two-base overhangs. Owing to the nature of its cleavage pattern, it is likely that MspI would represent a new structural class of restriction endonucleases. Crystals of the dimeric MspI restriction enzyme bound to a duplex DNA molecule containing the specific recognition sequence have been obtained by vapor-diffusion techniques in the presence of polyethylene glycol as precipitant. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 50.2, b = 131.6, c = 59.3 A, beta = 109.7 degrees. The crystals contain one dimeric complex in the asymmetric unit. A complete native data set has been collected to a resolution of 2.05 A by cryo-crystallographic methods, with an R(merge) of 4.0%.

Crystallization of restriction endonuclease BamHI with nonspecific DNA.

Restriction endonucleases show extraordinary specificity in distinguishing specific from nonspecific DNA sequences. A single basepair change within the recognition sequence results in over a million-fold loss in activity. To understand the basis of this sequence discrimination, it is just as important to study the nonspecific complex as the specific complex. We describe here the crystallization of restriction endonuclease BamHI with several nonspecific oligonucleotides. The 11-mer, 5'-ATGAATCCATA-3', yielded cocrystals with BamHI, in the presence of low salt, that diffracted to 1.9 A with synchrotron radiation. The cocrystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 114.8 A, b = 91.1 A, c = 66.4 A, alpha = 90 degrees, beta = 90 degrees, gamma = 90 degrees. This success in the cocrystallization of BamHI with a nonspecific DNA provides insights for future attempts at crystallization of other nonspecific DNA-protein complexes.

Identification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage.

Fenton chemistry [Fenton (1894) J. Chem. Soc. 65, 899-910] techniques were employed to identify the residues involved in metal binding located at the active sites of restriction endonucleases. This process uses transition metals to catalytically oxidize the peptide linkage that is in close proximity to the amino acid residues involved in metal ligation. Fe2+ was used as the redox-active transition metal. It was expected that Fe2+ would bind to the endonucleases at the Mg2+-binding site [Liaw et al. (1993) Biochemistry 32, 7999-4003; Ermácora et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387; Soundar and Colman (1993) J. Biol. Chem. 268, 5264-5271; Wei et al. (1994) Biochemistry 33, 7931-7936; Ettner et al. (1995) Biochemistry 34, 22-31; Hlavaty and Nowak (1997) Biochemistry 36, 15515-15525). Fe2+-mediated oxidation was successfully performed on TaqI endonulease, suggesting that this approach could be applied to a wide array of endonucleases [Cao and Barany (1998) J. Biol. Chem. 273, 33002-33010]. The restriction endonucleases BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, BsoBI, EcoRI, EcoRV, MspI, and HinP1I were subjected to oxidizing conditions in the presence of Fe2+ and ascorbate. All proteins were inactivated upon treatment with Fe2+ and ascorbate. BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, and BsoBI were specifically cleaved upon treatment with Fe2+/ascorbate. The site of Fe2+/ascorbate-induced protein cleavage for each enzyme was determined. The Fe2+-mediated oxidative cleavage of BamHI occurs between residues Glu77 and Lys78. Glu77 has been shown by structural and mutational studies to be involved in both metal ligation and catalysis [Newman et al. (1995) Science 269, 656-663; Viadiu and Aggarwal (1998) Nat. Struct. Biol. 5, 910-916; Xu and Schildkraut (1991) J. Biol. Chem. 266, 4425-4429]. The sites of Fe2+/ascorbate-induced cleavage for PvuII, FokI, BglI, and BsoBI agree with the metal-binding sites identified in their corresponding three-dimensional structures or from mutational studies [Cheng et al. (1994) EMBO J. 13, 3297-3935; Wah et al. (1997) Nature 388, 97-100; Newman et al. (1998) EMBO J. 17, 5466-5476; Ruan et al. (1997) Gene 188, 35-39]. The metal-binding residues of BglII, SfiI, and BssSI are proposed based on amino acid sequencing of their Fe2+/ascorbate-generated cleavage fragments. These results suggest that Fenton chemistry may be a useful methodology in identifying amino acids involved in metal binding in endonucleases.

Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution.

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1. 5 A resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar alpha/beta core, but in BglII, the core is augmented by a beta-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein-DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases.

Crystallization and preliminary diffraction analysis of the HincII restriction endonuclease-DNA complex.

Crystals of the 60 kDa dimeric HincII restriction enzyme bound to a 12 base-pair dyad-symmetric duplex DNA carrying the specific 5'-GTCGAC recognition site have been obtained. Crystals grew by hanging-drop vapor diffusion from solutions containing polyethylene glycol 4000 as precipitating agent. The rod-shaped crystals belong to space group I222 (or I2(1)2(1)2(1)), with unit-cell dimensions a = 66.9, b = 176.7, c = 256.0 A. There are most likely to be two dimeric complexes in the asymmetric unit. A complete native data set has been collected from a high-energy synchrotron source to a resolution of 2.5 A at 100 K, with an R(merge) of 4.8%.

Genetic analysis of the base-specific contacts of BamHI restriction endonuclease.

Here, we investigate the highly specific interaction of the BamHI endonuclease with its cognate recognition sequence GGATCC by determining which amino acid residues can be substituted at the DNA interface while maintaining specificity. Mutational studies, together with the structural determination of the restriction endonuclease BamHI have revealed the amino acid residues which are involved in DNA catalysis and those which play a role in the specific binding of the enzyme to its cognate DNA recognition sequence. Amino acid residues N116, S118, R122, D154 and R155 are involved in DNA sequence recognition and are located in the major groove in close proximity to the nucleotide bases comprising the recognition sequence. Cassette mutagenesis of these amino acids, together with in vivo transcriptional interference selection, was used to identify an array of substitutions which maintain site-specific binding to the cognate GGATCC sequence. This approach has demonstrated the extent of acceptable variation among amino acid residues which are directly involved in site-specific binding. One variant, double mutant N116H, S118G was found to cleave DNA only when the adenine base in the recognition site is methylated.

A mutant of BamHI restriction endonuclease which requires N6-methyladenine for cleavage.

Amino acid residues Asn116 and Ser118 of the restriction endonuclease BamHI make several sequence-specific and water-bridged contacts to the DNA bases. An in vivo selection was used to isolate BamHI variants at position 116, 118 and 122 which maintained sequence specificity to GGATCC sites. Here, the variants N116H, N116H/S118G and S118G were purified and characterized. The variants N116H and N116H/S118G were found to have lost their ability to cleave unmethylated GGATCC sequences by more than two orders of magnitude, while maintaining nearly wild-type levels of activity on the N6-methyladenine-containing sequence, GGmATCC. In contrast, wild-type BamHI and variant S118G have only a three- to fourfold lower activity on unmethylated GGATCC sequences compared with GGmATCC sequences. The N116 to H116 mutation has effectively altered the specificity of BamHI from an endonuclease which recognizes and cleaves GGATCC and GGmATC, to an endonuclease which only cleaves GGmATCC. The N116H change of specificity is due to the lowered binding affinity for the unmethylated sequence because of the loss of two asparagine-DNA hydrogen bonds and the introduction of a favorable van der Waals contact between the imidazole group of histidine and the N6-methyl group of adenine.

Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence.

The crystal structure of the type II restriction endonuclease BglI bound to DNA containing its specific recognition sequence has been determined at 2.2 A resolution. This is the first structure of a restriction endonuclease that recognizes and cleaves an interrupted DNA sequence, producing 3' overhanging ends. BglI is a homodimer that binds its specific DNA sequence with the minor groove facing the protein. Parts of the enzyme reach into both the major and minor grooves to contact the edges of the bases within the recognition half-sites. The arrangement of active site residues is strikingly similar to other restriction endonucleases, but the co-ordination of two calcium ions at the active site gives new insight into the catalytic mechanism. Surprisingly, the core of a BglI subunit displays a striking similarity to subunits of EcoRV and PvuII, but the dimer structure is dramatically different. The BglI-DNA complex demonstrates, for the first time, that a conserved subunit fold can dimerize in more than one way, resulting in different DNA cleavage patterns.

Structure of FokI has implications for DNA cleavage.

FokI is a member an unusual class of restriction enzymes that recognize a specific DNA sequence and cleave nonspecifically a short distance away from that sequence. FokI consists of an N-terminal DNA recognition domain and a C-terminal cleavage domain. The bipartite nature of FokI has led to the development of artificial enzymes with novel specificities. We have solved the structure of FokI to 2.3 A resolution. The structure reveals a dimer, in which the dimerization interface is mediated by the cleavage domain. Each monomer has an overall conformation similar to that found in the FokI-DNA complex, with the cleavage domain packing alongside the DNA recognition domain. In corroboration with the cleavage data presented in the accompanying paper in this issue of Proceedings, we propose a model for FokI DNA cleavage that requires the dimerization of FokI on DNA to cleave both DNA strands.

FokI dimerization is required for DNA cleavage.

FokI is a type IIs restriction endonuclease comprised of a DNA recognition domain and a catalytic domain. The structural similarity of the FokI catalytic domain to the type II restriction endonuclease BamHI monomer suggested that the FokI catalytic domains may dimerize. In addition, the FokI structure, presented in an accompanying paper in this issue of Proceedings, reveals a dimerization interface between catalytic domains. We provide evidence here that FokI catalytic domain must dimerize for DNA cleavage to occur. First, we show that the rate of DNA cleavage catalyzed by various concentrations of FokI are not directly proportional to the protein concentration, suggesting a cooperative effect for DNA cleavage. Second, we constructed a FokI variant, FokN13Y, which is unable to bind the FokI recognition sequence but when mixed with wild-type FokI increases the rate of DNA cleavage. Additionally, the FokI catalytic domain that lacks the DNA binding domain was shown to increase the rate of wild-type FokI cleavage of DNA. We also constructed an FokI variant, FokD483A, R487A, which should be defective for dimerization because the altered residues reside at the putative dimerization interface. Consistent with the FokI dimerization model, the variant FokD483A, R487A revealed greatly impaired DNA cleavage. Based on our work and previous reports, we discuss a pathway of DNA binding, dimerization, and cleavage by FokI endonuclease.

Structure of the multimodular endonuclease FokI bound to DNA.

FokI is a member of an unusual class of bipartite restriction enzymes that recognize a specific DNA sequence and cleave DNA nonspecifically a short distance away from that sequence. Because of its unusual bipartite nature, FokI has been used to create artificial enzymes with new specificities. We have determined the crystal structure at 2.8A resolution of the complete FokI enzyme bound to DNA. As anticipated, the enzyme contains amino- and carboxy-terminal domains corresponding to the DNA-recognition and cleavage functions, respectively. The recognition domain is made of three smaller subdomains (D1, D2 and D3) which are evolutionarily related to the helix-turn-helix-containing DNA-binding domain of the catabolite gene activator protein CAP. The CAP core has been extensively embellished in the first two subdomains, whereas in the third subdomain it has been co-opted for protein-protein interactions. Surprisingly, the cleavage domain contains only a single catalytic centre, raising the question of how monomeric FokI manages to cleave both DNA strands. Unexpectedly, the cleavage domain is sequestered in a 'piggyback' fashion by the recognition domain. The structure suggests a new mechanism for nuclease activation and provides a framework for the design of chimaeric enzymes with altered specificities.

Crystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA.

FokI is a type IIs restriction endonuclease which recognizes an asymmetric DNA sequence and cleaves DNA a short distance away from the sequence. The enzyme is bipartite in nature with its DNA recognition and cleavage functions located on distinct domains. We report here cocrystals of the complete FokI enzyme (579 amino acids) bound to a 20-bp DNA fragment containing its recognition sequence. The complex is amongst the largest protein-DNA complexes to be crystallized, and required macroseeding techniques for optimal crystal growth. The cocrystals diffract to at least 2.8 A in resolution and belong to space group P2(1) with unit cell dimensions of a=67.9 A, b=119.8 A, c=69.1 A, beta = 96.6 degrees. Using specific amino acid analysis we show that asymmetric unit contains a single FokI molecule bound to the 20-bp DNA fragment. This paper reports the first cocrystals of a type IIs restriction endonuclease.

Structure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding.

The crystal structure of restriction endonuclease Bam HI complexed to DNA has been determined at 2.2 angstrom resolution. The DNA binds in the cleft and retains a B-DNA type of conformation. The enzyme, however, undergoes a series of conformational changes, including rotation of subunits and folding of disordered regions. The most striking conformational change is the unraveling of carboxyl-terminal alpha helices to form partially disordered "arms." The arm from one subunit fits into the minor groove while the arm from the symmetry related subunit follows the DNA sugar-phosphate backbone. Recognition of DNA base pairs occurs primarily in the major groove, with a few interactions occurring in the minor groove. Tightly bound water molecules play an equally important role as side chain and main chain atoms in the recognition of base pairs. The complex also provides new insights into the mechanism by which the enzyme catalyzes the hydrolysis of DNA phosphodiester groups.

Crystal structure of the PvuII restriction endonuclease.

Crystal structures have now been determined for the R.PvuII restriction endonuclease as a protein-DNA complex [Cheng et al., EMBO J. 13 (1994) 3927-3935; this report] and in apo-form [Athanasiadis et al., Nature Struc. Biol. 1 (1994) 469-475; our unpublished result]. The structures indicate how the interaction with DNA might proceed [Riddihough, Nature 370 (1994) 78].

A model for DNA binding and enzyme action derived from crystallographic studies of the TaqI N6-adenine-methyltransferase.

The crystal structures of the DNA-N6-adenine-methyltransferase M.TaqI, in complexes with the cofactor S-adenosyl-L-methionine (AdoMet) and the competitive inhibitor sinefungin (Sf) show identical folding of the polypeptide chains into two domains. The N-terminal domain carries the cofactor-binding site, the C-terminal domain is thought to be implicated in sequence-specific DNA binding. Model building of the M.TaqI-DNA complex suggests that the adenine to be methylated swings out of the double helix as found previously in the cytosine-C5-MTase HhaI DNA co-crystal structure. A torsion of the methionine moiety of the cofactor is required to bring the methyl group within reach of the swung-out base and allow methyl group transfer.

Three-dimensional structure of the adenine-specific DNA methyltransferase M.Taq I in complex with the cofactor S-adenosylmethionine.

The Thermus aquaticus DNA methyltransferase M.Taq I (EC 2.1.1.72) methylates N6 of adenine in the specific double-helical DNA sequence TCGA by transfer of --CH3 from the cofactor S-adenosyl-L-methionine. The x-ray crystal structure at 2.4-A resolution of this enzyme in complex with S-adenosylmethionine shows alpha/beta folding of the polypeptide into two domains of about equal size. They are arranged in the form of a C with a wide cleft suitable to accommodate the DNA substrate. The N-terminal domain is dominated by a nine-stranded beta-sheet; it contains the two conserved segments typical for N-methyltransferases which form a pocket for cofactor binding. The C-terminal domain is formed by four small beta-sheets and alpha-helices. The three-dimensional folding of M.Taq I is similar to that of the cytosine-specific Hha I methyltransferase, where the large beta-sheet in the N-terminal domain contains all conserved segments and the enzymatically functional parts, and the smaller C-terminal domain is less structured.

Structure of PvuII endonuclease with cognate DNA.

We have determined the structure of PvuII endonuclease complexed with cognate DNA by X-ray crystallography. The DNA substrate is bound with a single homodimeric protein, each subunit of which reveals three structural regions. The catalytic region strongly resembles structures of other restriction endonucleases, even though these regions have dissimilar primary sequences. Comparison of the active site with those of EcoRV and EcoRI endonucleases reveals a conserved triplet sequence close to the reactive phosphodiester group and a conserved acidic pair that may represent the ligands for the catalytic cofactor Mg2+. The DNA duplex is not significantly bent and maintains a B-DNA-like conformation. The subunit interface region of the homodimeric protein consists of a pseudo-three-helix bundle. Direct contacts between the protein and the base pairs of the PvuII recognition site occur exclusively in the major groove through two antiparallel beta strands from the sequence recognition region of the protein. Water-mediated contacts are made in the minor grooves to central bases of the site. If restriction enzymes do share a common ancestor, as has been proposed, their catalytic regions have been very strongly conserved, while their subunit interfaces and DNA sequence recognition regions have undergone remarkable structural variation.

Crystallization and preliminary X-ray analysis of restriction endonuclease BamHI-DNA complex.

Restriction endonuclease BamHI from Bacillus amyloliquefaciens has been co-crystallized with a 12 bp DNA fragment that encompasses its recognition site. The co-crystals diffract to at least 1.95 A resolution and belong to space group P2(1)2(1)2(1). The unit cell parameters are a = 108.8 A, b = 81.9 A, c = 68.8 A, consistent with one complex in the crystallographic asymmetric unit. The direction of the DNA appears to be along the b axis. In order to achieve end to end stacking of DNA, the complex must lie on the screw axis along b. A self-rotation function has determined the directions of the non-crystallographic 2-fold axes.