Abstract
Homing endonucleases are highly specific DNA cleaving enzymes that are encoded within genomes of all forms of microbial life including phage and eukaryotic organelles. These proteins drive the mobility and persistence of their own reading frames. The genes that encode homing endonucleases are often embedded within self-splicing elements such as group I introns, group II introns and inteins. This combination of molecular functions is mutually advantageous: the endonuclease activity allows surrounding introns and inteins to act as invasive DNA elements, while the splicing activity allows the endonuclease gene to invade a coding sequence without disrupting its product. Crystallographic analyses of representatives from all known homing endonuclease families have illustrated both their mechanisms of action and their evolutionary relationships to a wide range of host proteins. Several homing endonucleases have been completely redesigned and used for a variety of genome engineering applications. Recent efforts to augment homing endonucleases with auxiliary DNA recognition elements and/or nucleic acid processing factors has further accelerated their use for applications that demand exceptionally high specificity and activity.
Highlights
Homing endonucleases, termed ‘meganucleases’, are highly specific DNA cleaving enzymes, found within all forms of microbial life as well as in eukaryotic mitochondria and chloroplasts, that are encoded by genes that display genetic mobility and persistence
Within 2 years, the complete nucleotide sequence of that mobile element, corresponding to a group I intron, was determined from several yeast strains. These analyses indicated that the intron was exceptionally long (1,143 base pairs), and contained an apparent reading frame that might encode a 235 residue protein [4]
Crystallographic structure analyses of all the redesigned enzymes in this study indicated that the basis of this behavior could be observed in patterns of structural context-dependence, extending across a local network of adjacent side chains and corresponding DNA base pairs, that caused unpredictable differences in DNA backbone conformation and side chains rotamers
Summary
The discovery of mobile introns and their homing endonucleases dates back to the 1970s. (i) Alteration of homing endonuclease target specificity at individual base pairs Early studies provided multiple examples where mutation of individual residues in a homing endonuclease DNAbinding surface resulted in a change in the specificity at a Figure 4 Redesign of a LAGLIDADG homing endonuclease (HE; termed a ‘meganuclease’) for a specific genome engineering application (such as modification of a disease-associated human gene locus) involves the alteration of a substantial fraction of its DNA-contacting residues, as well as further optimization of neighboring positions on the protein scaffold. Extremely informative reporter systems and assays that allow precise measurements and quantitation of the mechanisms, efficiency, and repair pathway choice and outcome(s) resulting from nuclease-induced double-strand breaks have been developed [133,134,135], facilitating the refinement and optimization of such systems for genome engineering applications
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