Zinc Finger Basics
ZINC FINGER BASICS
Zinc Finger Proteins
Zinc finger proteins are among the most abundant proteins in eukaryotic genomes with diversified functions like DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. (John H. Laity et al..).
Structure of Cys2His2 Zinc Finger Protein
Fig: Structure of Zinc Finger Protein, having a ²²± structure stabilized by the chelation of Zinc ion between the conserved Cys2His2 residues.
The Cys2His2 zinc finger proteins are a class of DNA-binding proteins that contain sequences of the form (Tyr, Phe)-Xaa-Cys-Xaa2-4-Cys-Xaa3-Phe-Xaa5-Leu-Xaa2-His-Xaa3-5-His, usually in tandem arrays (Yang-Gyun Kim et al,1995).
Each zinc finger motif consists of ~ 30 amino acids and folds into a ²²± -structure, which is stabilized by the chelation of a zinc ion between the two conserved cystine residues within the ² strand and two histidine residues within the ± helix ( Monika Pap worth et al..2005).
Mode of Binding of ZFP
Fig : A Two Finger Protein binding to a specific sequence of DNA. Amino acid residues from -1 to +6 make contact with the DNA.
Zinc Finger protein binds to the DNA helix by inserting ± helix into the major groove of the double helix. Each finger recognizes a specific triplet base in the DNA. Amino acid residues at positions -1, +1, +2, +3, +4, +5 and +6 relative to the start of the ±-helix determine the sequence specificity of the Zinc Finger motif. So, Zinc Finger motifs with varied sequence specificity can be obtained just by changing the key amino acid residues while maintaining the remaining amino acids as a consensus backbone (K. Kandavelou et al). Already ZF motifs recognizing various GNN, CNN and ANN triplets are identified (David J Segal et al, 2006). Zinc finger proteins recognizing long sequences of DNA can be obtained by linking appropriate Zinc finger motifs.(K.Kandavelou et al)
Zinc Finger Technology
These custom designed ZF proteins can be potentially fused to various effector domains like activators, repressors, nucleases to form chimeric regulatory entities with improved specificity (Monika Papworth et al, 2005).
Zinc Finger Nucleases And Genome Engineering
Zinc finger nucleases are custom designed nucleases that combine the specific DNA binding region of the Zinc finger proteins along with the non specific cleavage domain of an endonuclease. These chimeric nucleases which can recognise loger sequence of DNA and render a double stranded break at certain specific sites has shown the promise to play a crucial role in performing targeted genome engineering.
Double stranded nicks caused in a genome will induce homologous recombination as a natural means of repairing the break as they are harmful if left unrepaired. The repair occurs as a copy paste mechanism using the homologous DNA segment from the undamaged sister-chromatid as a template.
Targeted genome engineering can be achieved when the process of replacing a gene by HR, uses an extra chromosomal fragment of donor DNA and invokes the cell s own repair machinery for gene correction.
Requirements For DSB
In order to cause a double stranded break, these chimeric enzymes require dimerisation of the nuclease domain similar to the Fok1 restriction endonuclease from which the non specific cleavage domain was derived.
Two closely occuring inverted binding sites separated anywhere between 4 to16 bp can facilitate dimerisation and thereby be excellent targets for Zinc Finger nucleases. ( Mala Mani et al, 2005)
Fig: Two 9 bp recognition sites in an inverted orientation will cause dimerisation of the nuclease domain leading to a DSB
For a three finger protein to cause a double stranded break, it effectively requires two 9 bp recognition site oriented in a tail to tail fashion separated by a spacer. The dimerisation of the two Zinc finger nucleases will enable the recognition of a specific 18 bp sequence, which is long enough to specify a unique address in plants and mammalian genome. This proves the power of Zinc finger nucleases to make a unique cut in the eukaryotic genome.
Effective use of this powerful technology requires the judicious selection of binding sites for Zinc Finger Nuclease binding. Bind-Predict has been developed keeping this in mind.