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...

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.

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.

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...

BackgroundThe initiating nucleotide found at the 5’ end of primary transcripts has a distinctive triphosphorylated end that distinguishes these transcripts from all other RNA species. Recognizing this distinction is key to deconvoluting the primary transcriptome from the plethora of processed transcripts that confound analysis of the transcriptome. The currently available methods do not use targeted enrichment for the 5′end of primary transcripts, but rather attempt to deplete non-targeted RNA.ResultsWe developed a method, Cappable-seq, for directly enriching for the 5' end of primary transcripts and enabling determination of transcription start sites at single base resolution. This is achieved by enzymatically modifying the 5′ triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli, achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and identified TSS in a microbiome.ConclusionsCappable-seq allows for the first time the capture of the 5′ end of primary transcripts. This enables a unique robust TSS determination in bacteria and microbiomes.  In addition to and beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression in any microbiome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2539-z) contains supplementary material, which is available to authorized users.