Site-directed mutagenesis of gene sequences in cells of plants, Essay - 1

Site-directed mutagenesis of gene sequences in cells of plants, insects and animals can be achieved in situ used engineered zinc - Essay Example It is now well known that there are many naturally occurring zinc finger motifs, the most common of which is the Cys2His2 or C2H2 zinc finger. This particular motif is made up of 2 antiparallel ?-sheets and an ?-helix that are coordinated by 2 Histidine (his) and 2 Cysteine (cys) residues binding a zinc atom (Figure 1). This binding, plus an inner structural hydrophobic core, stabilizes the secondary structure of the protein. Figure 1. The classic zinc finger protein is made up of approximately 30 amino acids where two cysteine and two histidine residues bind to a zinc atom. The zinc finger proteins are known mediators of metabolic interactions including protein-protein interactions and RNA binding. However, the most known are their roles in DNA sequence-specific binding. From early studies on the classical C2H2 zinc finger, it was found that the zinc finger differed in principle from the other DNA-binding proteins because several zinc fingers can be linked linearly to recognize DNA sequences of varying lengths (Klug, 2005). In contrast, other DNA-binding proteins utilize the symmetry of the double helix. Usually, more than one zinc finger domain participates in binding the DNA. Each zinc finger domain binds to three nucleotide (nt) bases on the major groove of the DNA. The ?-helix, also known as the recognition helix, binds to three or more bases of with specific sequences on the DNA. Since zinc finger proteins could have more than one zinc finger motif, the DNA contacts of adjacent or arrayed zinc fingers could overlap (Iuchi, 2005). Regions that are bound by the zinc fingers are usually spaced at 3 – 5 bp apart. The modular design of the zinc fingers allows it to interact with the DNA at different regions, and allows them to be involved in many DNA interaction reactions. Thus, it is not surprising that the zinc fingers are the most encoded motif in the genome and are very abundant in nature. The highly sequence-specific DNA binding property of zinc fi ngers offered a lot of potential as a tool for regulating gene expression or for manipulating the genome. From the initial basic studies arose the design and engineering of zinc finger proteins for binding specific regions in the DNA, and synthesis of zinc finger nucleases for cutting the DNA at target sites and introduction of changes to the DNA (Davis & Stokoe, 2010; Durai, et. al, 2005). Zinc finger arrays can be engineered to bind long stretches of known regions in the genome and with the ultimate goal of finding cures to notable diseases. Some applications of zinc finger arrays are the inhibition of HIV-1 expression (Reynolds, et al., 2003) and the disruption of herpes simplex virus infection (Papworth, et al., 2003). To emphasize the importance of the zinc finger proteins, an online database was established that compiles information on known (naturally occurring) and engineered zinc fingers and zinc finger arrays (Fu, et al., 2009). Zinc Finger Nucleases A zinc finger nuclease (ZFN) is an engineered restriction enzyme that consists of a zinc finger array designed to recognize specific nucleotide sequences in the DNA, and a non-specific nuclease domain. Usually, the zinc fingers in the array have similar motifs as that of Cys2His2 zinc finger protein. The engineered zinc finger is fused with the cleavage domain of the restriction enzyme FokI endonuclease. Since FokI will cut the DNA only