Ira Schildkraut

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 2.8 Å 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.

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.

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 Å 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.

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 Å 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 α/β core, but in BglII, the core is augmented by a β-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.

Structure of restriction endonuclease BamHI phased at 1.95 å resolution by MAD analysis

BACKGROUND Type II restriction endonucleases recognize DNA sequences that vary between four to eight base pairs, and require only Mg2+ as a cofactor to catalyze the hydrolysis of DNA. Their protein sequences display a surprising lack of similarity, and no recurring structural motif analogous to the helix-turn-helix or the zinc finger of transcription factors, has yet been discovered. RESULTS We have determined the crystal structure of restriction endonuclease BamHI at 1.95 A resolution. The structure was solved by combining phase information derived from multi-wavelength X-ray data by algebraic and maximum likelihood methods. The BamHI subunit consists of a central beta-sheet with alpha-helices on both sides. The dimer configuration reveals a large cleft which could accommodate B-form DNA. Mutants of the enzyme that are deficient in cleavage are located at or near the putative DNA-binding cleft. BamHI and endonuclease EcoRI share a common core motif (CCM) consisting of five beta-strands and two helices. It remains to be determined if other restriction enzymes also contain the CCM. CONCLUSIONS The structure of BamHI provides the first clear evidence that there may be substantial structural homology amongst restriction enzymes, even though it is undetectable at the sequence level.

A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA.

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA. The structures reveal two different binding states of SfiI: one with both DNA‐binding sites fully occupied and the other with fully and partially occupied sites. These two states provide details on how SfiI recognizes and cleaves its target DNA sites, and gives insight into sequential binding events. The SfiI recognition sequence (GGCCNNNN↓NGGCC) is a subset of the recognition sequence of BglI (GCCNNNN↓NGGC), and both enzymes cleave their target DNAs to leave 3‐base 3′ overhangs. We show that even though SfiI is a tetramer and BglI is a dimer, and there is little sequence similarity between the two enzymes, their modes of DNA recognition are unusually similar.

Alteration of apparent restriction endonuclease recognition specificities by DNA methylases

An in vitro method of altering the apparent cleavage specificities of restriction endonucleases was developed using DNA modification methylases. This method was used to reduce the number of cleavage sites for 34 restriction endonucleases. In particular, single-site cleavages were achieved for Nhe I in Adeno-2 DNA and for Acc I and Hinc II in pBR322 DNA by specifically methylating all but one recognition sequence.

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.

Self-generated DNA termini relax the specificity of SgrAI restriction endonuclease

The primary target of SgrAI restriction endonuclease is a multiple sequence of the form 5′-CPu↓CCGGPyG. Previous work had indicated that SgrAI must bind two recognition sites simultaneously for catalysis [Bilcock, D. T., Daniels, L. E., Bath, A. J. & Halford, S. E. (1999) J. Biol. Chem. 274, 36379–36386]. In the present study, SgrAI is shown to cleave not only its canonical sequences, but also the sequences 5′-CPuCCGGPy(A,T,C) and 5′-CPuCCGGGG, both referred to as secondary sequences. On plasmid pSK7, SgrAI cleaves secondary sites 26-fold slower than the canonical site. However, the same plasmid, but without the canonical site, is cleaved 200-fold slower. We show that DNA termini generated by cleaving the canonical site for SgrAI assist in the cleavage of secondary sites. The SgrAI-termini in cis with respect to secondary site are markedly preferred over those in trans. The SgrAI-termini provided in a form of oligonucleotide duplex are also shown to stimulate canonical site cleavage. At a 40-fold molar excess of the SgrAI-termini over substrate, the SgrAI specificity is shown to improve by two orders of magnitude, because of concurrent 10-fold increase in the cleavage of canonical site and 50-fold decrease in the cleavage of secondary sites. The unconventional reaction pathway by which SgrAI utilizes the self-generated DNA termini to cleave its DNA targets has not been observed hitherto among type II restriction endonucleases. Based on our work and previous reports, a pathway of DNA binding and cleavage by the SgrAI restriction endonuclease is proposed.

M · FokI methylates adenine in both strands of its asymmetric recognition sequence

M.FokI, a type-IIS modification enzyme from Flavobacterium okeanokoites, was purified, and its activity was characterized in vitro. The enzyme was found to be a DNA-adenine methyltransferase and to methylate both strands of the asymmetric FokI recognition sequence: (formula; see text) M.FokI does not methylate single-stranded DNA, nor does it methylate double-stranded DNA at sequences other than FokI sites.