Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification

Abstract

Homing endonucleases are microbial DNA-cleaving enzymes that mobilize their own reading frames by generating double strand breaks at specific genomic invasion sites. These proteins display an economy of size, and yet recognize long DNA sequences (typically 20 to 30 base pairs). They exhibit a wide range of fidelity at individual nucleotide positions in a manner that is strongly influenced by host constraints on the coding sequence of the targeted gene. The activity of these proteins leads to site-specific recombination events that can result in the insertion, deletion, mutation, or correction of DNA sequences. Over the past fifteen years, the crystal structures of representatives from several homing endonuclease families have been solved, and methods have been described to create variants of these enzymes that cleave novel DNA targets. Engineered homing endonucleases proteins are now being used to generate targeted genomic modifications for a variety of biotech and medical applications.

Figure 1. Homing endonucleases and genetic homing

a: A mobile element consisting of a homing endonuclease gene (red bar) that is embedded within a self-splicing intron or intein (blue bars) resides within a host gene (grey bars). The homing endonuclease (red star) is expressed and cleaves a target site (green bar) that is found in a homologous allele of the host gene that lacks the entire element. The resulting double strand break is repaired by cellular machinery, generally leading either to repair via nonhomologous end-joining (not shown) or to repair via homologous recombination (HR). If HR successfully uses the intron-containing host allele (I+) as a corrective template, then the original uninterrupted allele (intron-minus; I−) is converted to an allele that now contains the intron and homing endonuclease gene (intron-plus; I+). b: A variety of biotechnology and gene therapy applications can potentially make use of the properties of a homing endonuclease, if such an enzyme is introduced into or expressed in a living cell, to drive a gene conversion process. Depending on the presence or absence (as well as the sequence) of a corrective DNA template for break repair, and on the catalytic properties of the endonuclease, such applications can lead to mutation, knockout, modification, or insertion of exogenous coding DNA into the gene target.

Figure 2. Homing endonuclease structural families

In all panels, divalent cations associated with the enzyme active sites are indicated by light green spheres. Bound zinc ions panel d are indicated by smaller dark green spheres. a: The phage-encoded HNH endonuclease I-HmuI (Shen et al., 2004) and GIY-YIG endonuclease I-TevI (VanRoey et al., 2002; VanRoey et al., 2001). These protein displays an extended, monomeric structure consisting of an N-terminal nuclease catalytic domain and a C-terminal DNA binding region that incorporates a C-terminal helix-turn-helix domain. The diagram of I-TevI is a composite of two separate crystal structures of the C-terminal region bount to DNA and the unbound catalytic domain. b: The cyanobacterial PD-(D/E)xK homing endonuclease I-Ssp6803I (a tetrameric intron-associated endonuclease that recognizes a target site in a tRNA host gene) (Zhao et al., 2007). c: The algal LAGLIDADG homing endonuclease I-CreI, which is a homodimer that recognizes a target site in the 23S rRNA encoding gene in the chloroplast genome of Chlamydomonas reinhardtii (Jurica et al., 1998). Similar endonucleases are encoded in fungal mitochondrial genomes and in archaea. These enzymes are found both as homodimers and as tandem repeats of two LAGLIDADG domains that form single chain monomeric proteins. d. The His-Cys box homing endonuclease I-PpoI, which is encoded in the nuclear genome in the slime mold Physarum polycephalum (Flick et al., 1998). This enzyme contains a variant of the HNH nuclease active site, and is therefore distantly related to the phage endonuclease I-HmuI (panel a), but the catalytic cores of these two enzymes have been incorporated into two very different structural scaffolds.

Figure 3. The DNA-binding surface and contacts formed by one subunit of the I-CreI homing endonuclease (from Figure 2c)

a: An antiparallel β-sheet structure presents a group of broadly distributed side chains to the major groove of the DNA target site, where they make a variety of specific and nonspecific contacts. b: A schematic of the contacts made by the structure shown in panel a. Hydrogen-bond acceptor and donor positions on each basepair are shown with concave and convex features on each base; direct contacts are shown with red arrows; water-mediated contacts are shown with blue arrows and blue spheres, which denote the water molecules in the crystal structure). Individual basepairs display a wide variation in the total number of contacts to protein atoms; this variation correlates approximately with the fidelity of recognition that is displayed at each position. Additional specificity is derived by indirect exploitation of sequence-specific conformational preferences of the DNA target, which is often bent as part of endonuclease binding and cleavage.

Figure 4. Evolution of homing endonucleases and disparate host gene products and functions from common nuclease ancestors

a: The GIY-YIG nuclease motif (center) has given rise both to phage-specific, monomeric homing endonucleases such as I-TevI (left) (VanRoey et al., 2002; VanRoey et al., 2001) and multimeric bacterial restriction endonucleases such as R.Eco29k (right) (Mak et al., 2010). The GIY-YIG catalytic motif in the core nuclease fold is shown in red in the upper panel. b: The LAGLIDADG fold (with its namesake catalytic motif shown in red in center panel) has given rise to monomeric and homodimeric homing endonucleases (such as the I-AniI endonuclease from Aspergillus nidulans, left) (Bolduc et al., 2003) and the broadly distributed DUF199 gene family, found in gram positive bacteria (right) (Kaiser et al., 2009; Knizewski and Ginalski, 2007). In this latter gene family, the LAGLIDADG scaffold is fused to a C-terminal helixturn-helix domain; members of this bacterial protein family (such as the WhiA protein from Streptomyces coelicolor) are thought to act as genetic regulators during processes such as sporulation (Ainsa et al., 2000).

Figure 5. Selections and direct redesign of homing endonuclease specificity

a: Several methods have been developed to assay the ability of mutated variants of homing endonucleases to recognize and cleave specific DNA target sites. These assays included the elimination of an integrated β-galactosidase gene in E. coli (top) (Seligman et al., 2002), the elimination of a cell death protein in E. coli (middle) (Doyon et al., 2006; Gruen et al., 2002) or the conversion of two inactive copies of a reporter gene into a single active copy, as a result of endonuclease-induced recombination in yeast (bottom) (Chames et al., 2005). As described in the literature, this latter assay has been incorporated in to a high-throughput screen where activity against a large number of potential target sites is measured in parallel for individual homing endonuclease variants (Arnould et al., 2006; Smith et al., 2006). A change in the position of a positive clone in the array of possible targets (shown by arrows pointing to two adjacent colonies with different sequences) demonstrates a shift in target site specificity. The basis of this latter assay, which directly measures endonuclease-induced recombination in a living cell, has been modified and used in many different eukaryotic cell contexts with a variety of reporters, particularly green fluorescent protein (GFP). b: Direct examination of a homing endonuclease-DNA bound cocrystal structure, often with the use of structure-based computational tools, can also be used to redirect protein-DNA contacts and corresponding specificity (Ashworth et al., 2006; Chevalier et al., 2003). Such methods can be used directly to redirect specificity at individual contact positions, or can be used to focus subsequent mutations to reduced numbers of positions and possible mutated amino acid identities.