A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

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.The type IIs restriction endonuclease FokI, isolated from Flavobacterium okeanokoites, recognizes an asymmetric nucleotide sequence and cleaves both DNA strands outside of the recognition site: 5Ј-GGATG(N) 9͞13 (1). The fokIRM genes have been cloned and sequenced (2, 3). The endonuclease consists of 587 aa with a molecular mass of 65.4 kDa (2, 4). FokI has been shown to exist as a monomer in solution, based on gel filtration and sedimentation experiments (4). It has been concluded that, when bound to DNA and analyzed by gel-mobility shift experiments, only one monomer of FokI was bound to its DNA recognition sequence (5). The gel-mobility shift experiments, where a 1:1 complex was observed (5), were performed with a FokI precleaved DNA. Therefore this complex represents FokI bound to the DNA product not to the DNA substrate.Unlike FokI, the typical type II restriction endonucleases, such as EcoRI or EcoRV, form a tight homodimer in solution and bind to DNA as a homodimer. Each monomeric subunit of homodimer contains one catalytic center. Mutational analysis by Waugh and Sauer (6) suggests that there is a single catalytic center per FokI monomer. This led them to conclude that either FokI must rearrange its catalytic center for sequential cleavage of each DNA strand or it must form a higher order complex to cleave both strands of DNA (6).Based on proteolytic studies, it was shown that FokI endonuclease contains two separate structural domains, one for DNA recognition and one for DNA cleavage (7). A purified 41-kDa N-terminal proteolytic fragment bound the recognition sequence specificall...

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 Å 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, from Flavobacterium okeanokoites, is a member of the unusual, type IIs class of restriction endonucleases that recognize a specific DNA sequence and cleave nonspecifically a short distance away from that sequence (1). FokI binds the cognate sequence 5Ј-GGATG-3Ј and cleaves DNA phosphodiester groups 9 bp away on this strand and 13 bp away on the complementary strand (Fig. 1). FokI has been shown to consist of two functionally distinct domains: an N-terminal DNA recognition domain and a C-terminal DNA cleavage domain (2). The modular nature of FokI has led to the development of artificial enzymes with new specificities (3-7).We recently reported the structure of FokI bound to a 20-bp DNA fragment containing the FokI cognate sequence (8). As expected, the protein has N-and C-terminal domains corresponding to the DNA recognition and cleavage functions, respectively. The recognition domain is comprised of three smaller subdomains (D1, D2, and D3) that are evolutionarily related to the helix-turn-helix-containing DNA-binding domain of the catabolite gene activator protein (9). The catabolite activator protein core has been embellished extensively in D1 and D2, whereas in D3 it has been co-opted for proteinprotein interactions. The cleavage domain is similar to a BamHI monomer and contains a single catalytic center, which raises the question of how monomeric FokI manages to cleave both strands. In a novel mechanism of nuclease activation, the recognition domain sequesters the cleavage domain through protein-protein interactions until its activity is required, whereby the cleavage domain dissociates from the recognition domain and swings over to the major groove for DNA cleavage. We have now determined the structure of FokI in the absence of DNA. The structure, determined at 2.3 Å resolution, 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...

SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of DNA activity and sequence specificity. Precleaved primary site DNA has been shown to be an allosteric effector [Hingorani-Varma & Bitinaite, (2003) J. Biol. Chem. 278, 40392-40399], stimulating cleavage of both primary (CR|CCGGYG, | indicates cut site, R=A,G, Y=C,T) and secondary (CR|CCGGY(A/C/T) and CR|CCGGGG) site DNA sequences. The fact that DNA is the allosteric effector of this endonuclease suggests at least two DNA binding sites on the functional SgrAI molecule, yet crystal structures of SgrAI [Dunten, et al., (2008) Nucleic Acids Res. 36, 5405–5416] show only one DNA duplex bound to one dimer of SgrAI. We show that SgrAI forms species larger than dimers or tetramers (High Molecular Weight Species, HMWS) in the presence of sufficient concentrations of SgrAI and its primary site DNA sequence, that are dependent on the concentration of the DNA bound SgrAI dimer. Analytical ultracentrifugation indicates that the HMWS is heterogeneous, has sedimentation coefficients of 15–20 s, and is composed of possibly 4–12 DNA bound SgrAI dimers. SgrAI bound to secondary site DNA will not form HMWS itself, but can bind to HMWS formed with primary site DNA and SgrAI. Uncleaved, as well as precleaved, primary site DNA is capable of stimulating HMWS formation. Stimulation of DNA cleavage by SgrAI, at primary as well as secondary sites, is also dependent on the concentration of primary site DNA (cleaved or uncleaved) bound SgrAI dimers. SgrAI bound to secondary site DNA does not have significant stimulatory activity. We propose that the oligomers of DNA bound SgrAI (i.e. HMWS) are the activated, or activatable, form of the enzyme.

Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the nicking and restriction sites are tailored to create ss extensions of custom sequence. The vector is then linearized by digestion with nicking and restriction endonucleases. To generate target molecules, a single deoxyuridine (dU) residue is placed 6–10 nt away from the 5′-end of each PCR primer. 5′ of dU the primer sequence is compatible either with an ss extension on the vector or with the ss extension of the next-in-line PCR product. After amplification, the dU is excised from the PCR products with the USER enzyme leaving PCR products flanked by 3′ ss extensions. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary ss extensions. By varying the design of the PCR primers, the protocol is easily adapted to perform one or more simultaneous DNA manipulations such as directional cloning, site-specific mutagenesis, sequence insertion or deletion and sequence assembly.