Cleaving DNA at Any Predetermined Site with Adapter-Primers and Class-IIS Restriction Enzymes

197

207

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...

mentioning

confidence: 99%

Structure of Fok I has implications for DNA cleavage

Wah

Bitinaite²,

Schildkraut³

et al. 1998

197

207

“…Indirect support for the presence of two domains comes from Szybalski et al (2,3). By combining Fok I with properly designed oligonucleotide adapters, they have cleverly devised a method that uses the separation between recognition site and endonuclease site to confer additional cleavage specificities (2,4,5).…”

mentioning

confidence: 99%

Functional domains in Fok I restriction endonuclease.

Chandrasegaran

1992

200

107

The PCR was used to alter transcriptionkl and translational signals surrounding the Flavobacterium okeanokoites restriction endonuclease (fokiR) gene, so as to achieve high expression in Eseherichia coli. By changing the ribosome-binding site sequence preceding the fokiR gene tO match the consensus E. coli signal and by placing a podive retroregulator stefla-loop sequence downstream of the gene, Fok I yield was increased to 5-8% of total cellular protein. Fok I was purified to homogeneity with phosphocellulose, DEAESephadex, and gel chromatography, yielding 50 mg of pure Fok I endonuclease per liter of culture medium. The recognition and cleavage domains of Fok I were analyzed by trypsin digestion. Fok I in the absence of a DNA substrate cleaves into a 58-kDa carboxyl-terminal and 8-kDa amino-terminal fngment. The 58-kDa fragment does not bind the DNA substrate. Fok I in the presence of a DNA substrate cleaves into a 41-kDa amino-terminal fragment and a 25-kDa carboxyl-terminal fragment. On further digestion, the 41-kDa fragment degrades into 30-kDa amino-terminal and 11-kDa carboxyl-terminal fragments. The cleaved fragments both bind DNA substrates, as does the 41-kDa fragment. Gel-mobility-shift assays indicate that all the protein contacts necessary for the sequence-speciftl recognition of DNA substrates are encoded within the 41-kDa fragment. Thus, the 41-kDa amino-terminal fragment constitutes the Fok I recognition domain. The 25-kDa fragment, purified by using a DEAE-Sephadex column, cleaves nonspecifically both methylated (pACYCfokIM) and nonmethylated (pTZ19R) DNA substrates in the presence of MgC12. Thus, the 25-kDa carboxyl-terminal fragment constitutes the Fok I cleavage domain.We have undertaken a detailed study of the Fok I restriction modification system from Flavobacterium okeanokoites. Fok

“…Indirect support for the presence of two domains within this enzyme comes from the work of Szybalski et al (2,3). By combining Fok I with properly designed oligonucleotide adapters, they have cleverly devised a method that uses the separation between the recognition site and the endonuclease site to confer additional cleavage specificities (2,4,5).…”

mentioning

confidence: 99%

Alteration of the cleavage distance of Fok I restriction endonuclease by insertion mutagenesis.

Chandrasegaran

1993

Fok I restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide 5'-GGATG-3' 5'-CATCC-3' in duplex DNA and cleaves 9 and 13 nucleotides away from the recognition site. Recently, we reported the presence of two distinct and separable protein domains within this enzyme-one for the sequence-specific recognition and the other for endonuclease activity. Here, we report the construction of two insertion mutants of Fok I endonuclease. The mutant enzymes were purified, and their cleavage properties were characterized. The mutants have the same DNA sequence specificity as the wild-type enzyme. However, compared with the wild-type enzyme, they cleave one nucleotide further away from the recognition site on both strands of the DNA substrates. Thus, it is possible to alter the cleavage distance of Fok I by protein engineering.We have undertaken a detailed study of the Fok I restrictionmodification system from Flavobacterium okeanokoites. Fok