The FokI nuclease is an enzyme isolated from the bacterium Flavobacterium okeanokoites. It cuts DNA molecules at specific locations. Its unique properties have established it as a valuable tool within molecular biology and genetic research.
Biochemical Properties and Structure
FokI is classified as a Type IIS restriction enzyme, a group characterized by their ability to recognize a specific DNA sequence but cleave the DNA at a separate, non-specific site some distance away from that recognition site. The enzyme is a relatively large protein, composed of 587 amino acids, which gives it a molecular mass of approximately 65.4 kilodaltons. Its structure is distinctly modular, featuring two independent functional regions.
It contains an N-terminal domain for DNA recognition and a C-terminal domain that performs the DNA cleavage. The DNA-binding domain precisely identifies an asymmetric five-base pair sequence, 5′-GGATG-3′, on the DNA strand. This recognition domain itself is intricate, comprising three smaller subdomains (D1, D2, and D3) that are structurally related to helix-turn-helix DNA-binding motifs found in other bacterial proteins. The C-terminal cleavage domain is not sequence-specific.
Mechanism of Action
The process begins when a single FokI molecule binds to its specific 5′-GGATG-3′ recognition sequence on a DNA strand. In its unbound state, the cleavage domain is sequestered by the recognition domain through protein-protein interactions. Upon DNA binding, a conformational change occurs within the enzyme, allowing the cleavage domain to dissociate from the recognition domain and swing into a position where it can interact with the DNA.
A single FokI molecule can bind DNA, but it cannot cleave it effectively as a monomer. DNA cutting activity requires the association of two FokI molecules, a process known as dimerization. This dimerization is mediated by the cleavage domains, which interact to form an active catalytic center. When two FokI monomers are bound to their respective recognition sites in close proximity, their cleavage domains pair up, often facilitated by the presence of divalent metal ions such as magnesium.
Once the active dimeric complex is formed, the combined nuclease domains induce a double-strand break in the DNA. This cut occurs at precise distances from the recognition site: 9 base pairs downstream on the strand containing the 5′-GGATG-3′ motif and 13 base pairs downstream on the complementary strand. This staggered cut produces a 4-base pair single-stranded overhang, commonly referred to as a sticky end.
Applications in Genome Engineering
The unique modularity of FokI has been harnessed by scientists to develop powerful tools for targeted genome engineering. FokI’s non-specific DNA cleavage domain can be separated from its natural DNA-binding domain and then fused to other, custom-designed DNA-binding proteins. This allows the nuclease activity to be directed to virtually any desired DNA sequence within a complex genome. The most notable examples of this technology are Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs).
Zinc Finger Nucleases (ZFNs)
ZFNs are artificial enzymes created by fusing the FokI cleavage domain to engineered zinc finger proteins (ZFPs). Zinc fingers are natural protein motifs, and specific arrangements of these motifs can be designed to recognize sequences ranging from 9 to 18 base pairs. Two separate ZFN monomers are typically designed to bind to opposite strands of a target DNA sequence. These two ZFNs bind in an inverted tail-to-tail orientation, separated by a specific spacer sequence, often 5 to 7 base pairs in length, which positions the fused FokI domains to dimerize and induce a precise double-strand break.
Transcription Activator-Like Effector Nucleases (TALENs)
Similarly, TALENs are constructed by fusing the FokI cleavage domain to Transcription Activator-Like Effector (TALE) proteins. TALE proteins have a unique repeating structure, where each repeat recognizes a single DNA base pair, enabling highly specific DNA targeting. Like ZFNs, two TALEN constructs are designed to bind to opposing DNA strands surrounding a target site, with a spacer in between. This arrangement ensures that the FokI nuclease domains are brought into close proximity, allowing them to dimerize and create a double-strand break at the intended genomic location. These induced double-strand breaks activate cellular DNA repair pathways, which can be manipulated to introduce specific genetic modifications.
Role in Modern Molecular Biology
FokI-based genome engineering tools, including ZFNs and TALENs, have been important in the development of targeted genetic manipulation. These systems rely on protein-based DNA recognition to guide the FokI nuclease. This mechanism contrasts with the RNA-guided approach employed by the more recently developed and widely known CRISPR-Cas9 system, which uses a small RNA molecule to direct its nuclease activity.
Despite the broad adoption of CRISPR-Cas9, FokI’s unique properties continue to be leveraged, particularly to enhance the specificity of other genome editing platforms. For example, the FokI cleavage domain has been fused to a catalytically inactive Cas9 (dCas9) to create hybrid nucleases. These dCas9-FokI fusions require two separate complexes, each guided by an RNA, to bind to nearby target sites, typically separated by 15 to 25 base pairs. This dual-targeting strategy requires two FokI domains to dimerize for DNA cleavage, which significantly reduces unintended off-target cutting. This approach provides a valuable alternative for high-precision genome editing in specific research contexts and therapeutic applications where minimizing off-target effects is a primary concern.