Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI

Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.     

Isolation and characterization of new homing endonuclease specificities at individual target site positions

Homing endonucleases are highly specific DNA endonucleases, encoded within mobile introns or inteins, that induce targeted recombination, double-strand repair and gene conversion of their cognate target sites. Due to their biological function and high level of target specificity, these enzymes are under intense investigation as tools for gene targeting. These studies require that naturally occurring enzymes be redesigned to recognize novel target sites. Here, we report studies in which the homodimeric LAGLIDADG homing endonuclease I-CreI is altered at individual side-chains corresponding to contact points to distinct base-pairs in its target site. The resulting enzyme constructs drive specific elimination of selected DNA targets in vivo and display shifted specificities of DNA binding and cleavage in vitro. Crystal structures of two of these constructs demonstrate that substitution of individual side-chain/DNA contact patterns can occur with almost no structural deformation or rearrangement of the surrounding complex, facilitating an isolated, modular redesign strategy for homing endonuclease activity and specificity.     

DNA binding and cleavage by the HNH homing endonuclease I-HmuI

The structure of I-HmuI, which represents the last family of homing endonucleases without a defining crystallographic structure, has been determined in complex with its DNA target. A series of diverse protein structural domains and motifs, contacting sequential stretches of nucleotide bases, are distributed along the DNA target. I-HmuI contains an N-terminal domain with a DNA-binding surface found in the I-PpoI homing endonuclease and an associated HNH/N active site found in the bacterial colicins, and a C-terminal DNA-binding domain previously observed in the I-TevI homing endonuclease. The combination and exchange of these features between protein families indicates that the genetic mobility associated with homing endonucleases extends to the level of independent structural domains. I-HmuI provides an unambiguous structural connection between the His-Cys box endonucleases and the bacterial colicins, supporting the hypothesis that these enzymes diverged from a common ancestral nuclease.     

The crystal structure of the gene targeting homing endonuclease I-SceI reveals the origins of its target site specificity

The I-SceI homing endonuclease enhances gene targeting by introducing double-strand breaks at specific chromosomal loci, thereby increasing the recombination frequency. Here, we report the crystal structure of the enzyme complexed to its DNA substrate and Ca(2+) determined at 2.25A resolution. The structure shows the prototypical beta-saddle of LAGLIDADG homing endonucleases that is contributed by two pseudo-symmetric domains. The high specificity of I-SceI is explained by the large number of protein-DNA contacts, many that are made by a long beta-hairpin loop that reaches into the major groove of the DNA. The DNA minor groove is compressed at the catalytic center, bringing the two scissile phosphodiester bonds into close proximity. The protein-Ca(2+)-DNA structure shows the protein bound to its DNA substrate in a pre-reactive state that is defined by the presence of two asymmetric active sites, one of which appears poised to first cleave the DNA bottom strand.     

Evolution of I-SceI homing endonucleases with increased DNA recognition site specificity

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G₊₄ base pair for the wild-type A:T₊₄ base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T₊₄ were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T₊₄ or the C:G₊₄ base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G₊₄ recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T₊₄ target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G₊₄ target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed ∼36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G₊₄ substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.     

Creation of an artificial bifunctional intein by grafting a homing endonuclease into a mini-intein

The majority of inteins are comprised of a protein splicing domain and a homing endonuclease domain. Experimental evidence has demonstrated that the splicing domain and the endonuclease domain in a bifunctional intein are largely independent of each other with respect to both structure and activity. Here, an artificial bifunctional intein has been created through the insertion of an existing homing endonuclease into a mini-intein that is naturally lacking this functionality. The gene for I-CreI, an intron-encoded homing endonuclease, was grafted into the monofunctional Mycobacterium xenopi GyrA intein at the putative site of the missing endonuclease. The resulting fusion protein was found to be capable of protein splicing similar to that of the parent intein. In addition, the protein demonstrated site-specific endonuclease activity that is characteristic of the I-CreI homing endonuclease. The function of each domain therefore remained unaffected by the presence of the other domain. This artificial fusion of the two domains is a potential novel mobile genetic element.     

DNA recognition by the homing endonuclease PI-SceI involves a divalent metal ion cofactor-induced conformational change

PI-SceI, a homing endonuclease of the LAGLIDADG family, consists of two domains involved in DNA cleavage and protein splicing, respectively. Both domains cooperate in binding the recognition sequence. Comparison of the structures of PI-SceI in the absence and presence of substrate reveals major conformational changes in both the protein and DNA. Notably, in the protein-splicing domain the loop comprising residues 53-70 and adopts a "closed" conformation, thus enabling it to interact with the DNA. We have studied the dynamics of DNA binding and subsequent loop movement by fluorescence techniques. Six amino acids in loop53-70 were individually replaced by cysteine and modified by fluorescein. The interaction of the modified PI-SceI variants with the substrate, unlabeled or labeled with tetramethylrhodamine, was analyzed in equilibrium and stopped-flow experiments. A kinetic scheme was established describing the interaction between PI-SceI and DNA. It is noteworthy that the apparent hinge-flap motion of loop53-70 is only observed in the presence of a divalent metal ion cofactor. Substitution of the major Mg2+-binding ligands in PI-SceI, Asp-218 and Asp-326, by Asn or "nicking" PI-SceI with trypsin at Arg-277, which interferes with formation of an active enzyme.substrate complex, both prevent the conformational change of loop53-70. Deletion of the loop inactivates the enzyme. We conclude that loop53-70 is an important structural element that couples DNA recognition by the splicing domain with DNA cleavage by the catalytic domain and as such "communicates" with the Mg2+ binding sites at the catalytic centers.     

DNA recognition by the EcoRV restriction endonuclease probed using base analogues

The EcoRV restriction endonuclease recognises palindromic GATATC sequences and cuts between the central T and dA bases in a reaction that has an absolute requirement for a divalent metal ion, physiologically Mg(2+). Use has been made of base analogues, which delete hydrogen bonds between the protein and DNA (or hydrophobic interactions in the case of the 5-CH(3) group of thymine), to evaluate the roles of the outer two base-pairs (GATATC) in DNA recognition. Selectivity arises at both the binding steps leading to the formation of the enzyme-DNA-metal ion ternary complex (assayed by measuring the dissociation constant in the presence of the non-reactive metal Ca(2+)) and the catalytic step (evaluated using single-turnover hydrolysis in the presence of Mg(2+)), with each protein-DNA contact contributing to recognition. With the A:T base-pair, binding was reduced by the amount expected for the simple loss of a single contact; much more severe effects were observed with the G:C base-pair, suggesting additional conformational perturbation. Most of the modified bases lowered the rate of hydrolysis; furthermore, the presence of an analogue in one strand of the duplex diminished cutting at the second, unmodified strand, indicative of communication between DNA binding and the active site. The essential metal ion Mg(2+) plays a key role in mediating interactions between the DNA binding site and active centre and in many instances rescue of hydrolysis was seen with Mn(2+). It is suggested that contacts between the GATATC site are required for tight binding and for the correct assembly of metal ions and bound water at the catalytic site, functions important in providing acid/base catalysis and transition state stabilisation.     

Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets

The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.     

Directed evolution of restriction endonuclease BstYI to achieve increased substrate specificity

Restriction endonucleases have proven to be especially resistant to engineering altered substrate specificity, in part, due to the requirement of a cognate DNA methyltransferase for cellular DNA protection. The thermophilic restriction endonuclease BstYI recognizes and cleaves all hexanucleotide sequences described by 5'-R GATCY-3' (where R=A or G and Y=C or T). The recognition of a degenerate sequence is a relatively common feature of the more than 3000 characterized restriction endonucleases. However, very little is known concerning substrate recognition by such an enzyme. Our objective was to investigate the substrate specificity of BstYI by attempting to increase the specificity to recognition of only AGATCT. By a novel genetic selection/screening process, two BstYI variants were isolated with a preference for AGATCT cleavage. A fundamental element of the selection process is modification of the Escherichia coli host genomic DNA by the BglII N4-cytosine methyltransferase to protect AGATCT sites. The amino acid substitutions resulting in a partial change of specificity were identified and combined into one superior variant designated NN1. BstYI variant NN1 displays a 12-fold preference for cleavage of AGATCT over AGATCC or GGATCT. Moreover, cleavage of the GGATCC sequence is no longer detected. This study provides further evidence that laboratory evolution strategies offer a powerful alternative to structure-guided protein design.     

Structure of a hyperthermophilic archaeal homing endonuclease, I-Tsp061I: contribution of cross-domain polar networks to thermostability

A novel LAGLIDADG-type homing endonuclease (HEase), I-Tsp061I, from the hyperthermophilic archaeon Thermoproteus sp. IC-061 16 S rRNA gene (rDNA) intron was characterized with respect to its structure, catalytic properties and thermostability. It was found that I-Tsp061I is a HEase isoschizomer of the previously described I-PogI and exhibits the highest thermostability among the known LAGLIDADG-type HEases. Determination of the crystal structure of I-Tsp061I at 2.1 A resolution using the multiple isomorphous replacement and anomalous scattering method revealed that the overall fold is similar to that of other known LAGLIDADG-type HEases, despite little sequence similarity between I-Tsp061I and those HEases. However, I-Tsp061I contains important cross-domain polar networks, unlike its mesophilic counterparts. Notably, the polar network Tyr6-Asp104-His180-107O-HOH12-104O-Asn177 exists across the two packed alpha-helices containing both the LAGLIDADG catalytic motif and the GxxxG hydrophobic helix bundle motif. Another important structural feature is the salt-bridge network Asp29-Arg31-Glu182 across N and C-terminal domain interface, which appears to contribute to the stability of the domain/domain packing. On the basis of these structural analyses and extensive mutational studies, we conclude that such cross-domain polar networks play key roles in stabilizing the catalytic center and domain packing, and underlie the hyperthermostability of I-Tsp061I.     

Coevolution of a homing endonuclease and its host target sequence

We have determined the specificity profile of the homing endonuclease I-AniI and compared it to the conservation of its host gene. Homing endonucleases are encoded within intervening sequences such as group I introns. They initiate the transfer of such elements by cleaving cognate alleles lacking the intron, leading to their transfer via homologous recombination. Each structural homing endonuclease family has arrived at an appropriate balance of specificity and fidelity that avoids toxicity while maximizing target recognition and invasiveness. I-AniI recognizes a strongly conserved target sequence in a host gene encoding apocytochrome B and has fine-tuned its specificity to correlate with wobble versus nonwobble positions across that sequence and to the amount of degeneracy inherent in individual codons. The physiological target site in the host gene is not the optimal substrate for recognition and cleavage: at least one target variant identified during a screen is bound more tightly and cleaved more rapidly. This is a result of the periodic cycle of intron homing, which at any time can present nonoptimal combinations of endonuclease specificity and insertion site sequences in a biological host.     

Profile of the DNA recognition site of the archaeal homing endonuclease I-DmoI.

I- Dmo I is a homing enzyme of the LAGLI-DADG type that recognizes up to 20 bp of DNA and is encoded by an archaeal intron of the hyperthermophilic archaeon Desulfurococcus mobilis . A combined mutational and DNA footprinting approach was employed to investigate the specificity of the I- Dmo I-substrate interaction. The results indicate that the enzyme binds primarily to short base paired regions that border the sites of DNA cleavage and intron insertion. The minimal substrate spans no more than 15 bp and while sequence degeneracy is tolerated in the DNA binding regions, the sequence and size of the cleavage region is highly conserved. The enzyme has a slow turnover rate and cuts the coding strand with a slight preference over the non-coding strand. Complex formation produces some distortion of the DNA double helix within the cleavage region. The data are compatible with the two DNA-binding domains of I- Dmo I bridging the minor groove, where cleavage occurs, and interacting within the major groove on either side, thereby stabilizing a distorted DNA double helix. This may provide a general mode of DNA interaction at least for the LAGLIDADG-type homing enzymes.     

In vivo analysis of the relationships between the splicing and homing activities of a group I intron-encoded I-ScaI/bi2-maturase of Saccharomyces capensis produced in the yeast cytoplasm

The I-ScaI/bi2-maturase of Saccharomyces capensis acts as a specific homing endonuclease promoting intron homing, and as a maturase promoting intron splicing. Using the universal code equivalent of the mitochondrial gene encoding the I-ScaI/bi2-maturase, a number of truncated forms of the synthetic gene were constructed, shortened on either side, as were several mutated alleles of the protein. The shortest translation product that fully retains both activities in vivo corresponds to 228 codons of the C-terminal region of the bi2 intron-encoded protein, whereas proteins resulting from more extensive deletions either at the N-terminus or at the C-terminus (up to 73 and four residues, respectively) were able to complement wholly the lack of endogenous maturase, but all lost the endonuclease activity. Similarly, all introduced mutations completely abolished the I-ScaI activity while some mutant proteins retained substantial splicing function. Immunodetection experiments demonstrated that different cytoplasmically translated forms of the I-ScaI/bi2-maturase protein were imported into mitochondria and correctly processed. They appeared to be tightly associated with mitochondrial membranes. Homology modelling of the I-ScaI/bi2-maturase protein allowed us to relate both enzymatic activities to elements of enzyme structure.     

Crystallization and preliminary X-ray studies of I-PpoI: a nuclear, intron-encoded homing endonuclease from Physarum polycephalum.

The homing endonuclease I-PpoI is encoded by an optional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26S rRNA gene. This endonuclease promotes the lateral transfer or "homing" of its encoding intron by recognizing and cleaving a partially symmetric, 15 bp homing site in 26S rDNA alleles that lack the Pp LSU 3 intron. The open reading frame encoding I-PpoI has been subcloned, and the endonuclease has been overproduced in E. coli. Purified recombinant I-PpoI has been co-crystallized with a 21 bp homing site DNA duplex. The crystals belong to space group P3(1)21, with unit cell dimensions a = b = 114 A, c = 89 A. The results of initial X-ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 A3/dalton. These experiments also provide strong evidence that I-PpoI contains several bound zinc ions as part of its structure.     

Characterization of intein homing endonuclease encoded in the DNA polymerase gene of Thermococcus marinus

The DNA polymerase gene of Thermococcus marinus ( Tma ) contains an intein inserted at the pol-b site that possesses a 1611-bp ORF encoding a 537-amino acid residue. The LAGLIDADG motif, often found in site-specific DNA endonucleases, was detected within the amino acid sequence of the intein. The intein endonuclease, denoted as PI- Tma , was purified as a naturally spliced product from the expression of the complete DNA polymerase gene in Escherichia coli . PI- Tma cleaved intein-less DNA sequences, leaving four-base-long, 3'-hydroxyl overhangs with 5'-phosphate. Nonpalindromic recognition sequences 19 bp long were also identified using partially complementary oligonucleotide pair sequences inserted into the plasmid pET-22b(+). Cleavage by PI- Tma was optimal when present in 50 mM glycine–NaOH (pH 10.5), 150 mM KCl and 12 mM MgCl₂ at 70 °C.     

Characterization of the cleavage site and the recognition sequence of the I-CreI DNA endonuclease encoded by the chloroplast ribosomal intron of Chlamydomonns reinhardtii

Summary The chloroplast ribosomal intron of Chlamydomonas reinhardtii encodes a sequence-specific DNA endonuclease (I-CreI), which is most probably involved in the mobility of this intron. Here we show that I-CreI generates a 4 by staggered cleavage just downstream of the intron insertion site. The I-CreI recognition sequence is 19–24 by in size and is located asymmetrically around the intron insertion site. Screening of natural variants of the I-CreI recognition sequence indicates that the I-CreI endonuclease tolerates single and even multiple base changes within its recognition sequence.     

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.