Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family

Novel family of putative homing endonuclease genes was recently discovered during analyses of metagenomic and genomic sequence data. One such protein is encoded within a group I intron that resides in the recA gene of the Bacillus thuringiensis 03058-36 bacteriophage. Named I-Bth0305I, the endonuclease cleaves a DNA target in the uninterrupted recA gene at a position immediately adjacent to the intron insertion site. The enzyme displays a multidomain, homodimeric architecture and footprints a DNA region of ~60 bp. Its highest specificity corresponds to a 14-bp pseudopalindromic sequence that is directly centered across the DNA cleavage site. Unlike many homing endonucleases, the specificity profile of the enzyme is evenly distributed across much of its target site, such that few single base pair substitutions cause a significant decrease in cleavage activity. A crystal structure of its C-terminal domain confirms a nuclease fold that is homologous to very short patch repair (Vsr) endonucleases. The domain architecture and DNA recognition profile displayed by I-Bth0305I, which is the prototype of a homing lineage that we term the 'EDxHD' family, are distinct from previously characterized homing endonucleases.     

Mining endonuclease cleavage determinants in genomic sequence data

Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold. Many of these grafts exhibited novel and unexpected specificities. These findings show that the information present in genomic data can be exploited for endonuclease specificity redesign.     

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.     

Adaptation of intronic homing endonuclease for successful horizontal transmission

Group I introns are thought to be self-propagating mobile elements, and are distributed over a wide range of organisms through horizontal transmission. Intron invasion is initiated through cleavage of a target DNA by a homing endonuclease encoded in an open reading frame (ORF) found within the intron. The intron is likely of no benefit to the host cell and is not maintained over time, leading to the accumulation of mutations after intron invasion. Therefore, regular invasional transmission of the intron to a new species at least once before its degeneration is likely essential for its evolutionary long-term existence. In many cases, the target is in a protein-coding region which is well conserved among organisms, but contains ambiguity at the third nucleotide position of the codon. Consequently, the homing endonuclease might be adapted to overcome sequence polymorphisms at the target site. To address whether codon degeneracy affects horizontal transmission, we investigated the recognition properties of a homing enzyme, I-CsmI, that is encoded in the intronic ORF of a group I intron located in the mitochondrial COB gene of the unicellular green alga Chlamydomonas smithii. We successfully expressed and purified three types of N-terminally truncated I-CsmI polypeptides, and assayed the efficiency of cleavage for 81 substrates containing single nucleotide substitutions. We found a slight but significant tendency that I-CsmI cleaves substrates containing a silent or tolerated amino acid change more efficiently than nonsilent or nontolerated ones. The published recognition properties of I-SpomI, I-ScaI, and I-SceII were reconsidered from this point of view, and we detected proficient adaptation of I-SpomI, I-ScaI, and I-SceII for target site sequence degeneracy. Based on the results described above, we propose that intronic homing enzymes are adapted to cleave sequences that might appear at the target region in various species, however, such adaptation becomes less prominent in proportion to the time elapsed after intron invasion into a new host.     

Evolutionary Dynamics of the mS952 Intron: A Novel Mitochondrial Group II Intron Encoding a LAGLIDADG Homing Endonuclease Gene

Examination of the mitochondrial small subunit ribosomal RNA (rns) gene of five species of the fungal genus Leptographium revealed that the gene has been invaded at least once at position 952 by a group II intron encoding a LAGLIDADG homing endonuclease gene. Phylogenetic analyses of the intron and homing endonuclease sequences indicated that each element in Leptographium species forms a single clade and is closely related to the group II intron/homing endonuclease gene composite element previously reported at position 952 of the mitochondrial rns gene of Cordyceps species and of Cryphonectria parasitica. The results of an intron survey of the mt rns gene of Leptographium species superimposed onto the phylogenetic analysis of the host organisms suggest that the composite element was transmitted vertically in Leptographium lundbergii. However, its stochastic distribution among strains of L. wingfieldii, L. terebrantis, and L. truncatum suggests that it has been horizontally transmitted by lateral gene transfer among these species, although the random presence of the intron may reflect multiple random loss events. A model is proposed describing the initial invasion of the group II intron in the rns gene of L. lundbergii by a LAGLIDADG homing endonuclease gene and subsequent evolution of this gene to recognize a novel DNA target site, which may now promote the mobility of the intron and homing endonuclease gene as a composite element.     

Functionally distinct nucleic acid binding sites for a group I intron encoded RNA maturase/DNA homing endonuclease

A large number of group I introns encode a family of homologous proteins that either promote intron splicing (maturases) or are site-specific DNA endonucleases that function in intron mobility (a process called "homing"). Genetic studies have shown that some of these proteins have both activities, yet how a single protein carries out both functions remains obscure. The similarity between respective DNA-binding sites and the RNA structure near the 5' and 3' splice sites has fueled speculation that such proteins may use analogous interactions to perform both functions. The Aspergillus nidulans mitochondrial COB group I intron encodes a bi-functional protein, I-AniI, that has both RNA maturase and site-specific DNA endonuclease activities in vitro. Here, we show that I-AniI shows distinctive features of the endonuclease family to which it belongs, including highly specific, tight binding and sequential DNA strand cleavage. Competition experiments demonstrate that I-AniI binds the COB intron RNA even in saturating concentrations of its DNA target site substrate, suggesting that the protein has a separate binding site for RNA. In addition, we provide evidence that two different DNA-binding site mutants of I-AniI have little effect on the protein's RNA maturation activity. Since RNA splicing is likely a secondary adaptation of the protein, these observations support a model in which homing endonucleases may have developed maturase function by utilizing a hitherto "non-functional" protein surface.     

Recognition of a common rDNA target site in archaea and eukarya by analogous LAGLIDADG and His-Cys box homing endonucleases

The presence of a homing endonuclease gene (HEG) within a microbial intron or intein empowers the entire element with the ability to invade genomic targets. The persistence of a homing endonuclease lineage depends in part on conservation of its DNA target site. One such rDNA sequence has been invaded both in archaea and in eukarya, by LAGLIDADG and His–Cys box homing endonucleases, respectively. The bases encoded by this target include a universally conserved ribosomal structure, termed helix 69 (H69) in the large ribosomal subunit. This region forms the ‘B2a’ intersubunit bridge to the small ribosomal subunit, contacts bound tRNA in the A- and P-sites, and acts as a trigger for ribosome disassembly through its interactions with ribosome recycling factor. We have determined the DNA-bound structure and specificity profile of an archaeal LAGLIDADG homing endonuclease (I-Vdi141I) that recognizes this target site, and compared its specificity with the analogous eukaryal His–Cys box endonuclease I-PpoI. These homodimeric endonuclease scaffolds have arrived at similar specificity profiles across their common biological target and analogous solutions to the problem of accommodating conserved asymmetries within the DNA sequence, but with differences at individual base pairs that are fine-tuned to the sequence conservation of archaeal versus eukaryal ribosomes.     

From monomeric to homodimeric endonucleases and back: engineering novel specificity of LAGLIDADG enzymes

Monomeric homing endonucleases of the LAGLIDADG family recognize DNA in a bipartite manner, reflecting the underlying structural assembly of two protein domains (A and B) related by pseudo 2-fold symmetry. This architecture allows for changes in DNA specificity via the distinct combination of these half-site domains. The key to engineering such hybrid proteins lies in the LAGLIDADG two-helix bundle that forms both the domain interface and the endonuclease active site. In this study, we utilize domain A of the monomeric I-DmoI to demonstrate the feasibility of generating functional homodimeric endonucleases that recognize palindromic DNA sequences derived from the original, non-palindromic target. Wild-type I-DmoI domain A is capable of forming a homodimer (H-DmoA) that binds tightly to, but does not cleave efficiently, its anticipated DNA target. Partial restoration of DNA cleavage ability was obtained by re-engineering the LAGLIDADG dimerization interface (H-DmoC). Upon fusing two copies of H-DmoC via a short peptide linker, a novel, site-specific DNA endonuclease was created (H-DmoC2). Like I-DmoI, H-DmoC2 is thermostable and cleaves the new target DNA to generate the predicted 4 nt 3'-OH overhangs but, unlike I-DmoI, H-DmoC2 retains stringent cleavage specificity when substituting Mn2+ for Mg2+ as co-factor. This novel endonuclease allows speculation regarding specificity of monomeric LAGLIDADG proteins, while it supports the evolutionary genesis of these proteins by a gene duplication event.     

In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional group I intron-encoded protein, I-AniI.

The AnCOB group I intron from Aspergillus nidulans encodes a homing DNA endonuclease called I-AniI which also functions as a maturase, assisting in AnCOB intron RNA splicing. In this investigation we biochemically characterized the endonuclease activity of I-AniI in vitro and utilized competition assays to probe the relationship between the RNA- and DNA-binding sites. Despite functioning as an RNA maturase, I-AniI still retains several characteristic properties of homing endonucleases including relaxed substrate specificity, DNA cleavage product retention and instability in the reaction buffer, which suggest that the protein has not undergone dramatic structural adaptations to function as an RNA-binding protein. Nitrocellulose filter binding and kinetic burst assays showed that both nucleic acids bind I-AniI with the same 1 : 1 stoichiometry. Furthermore, in vitro competition activity assays revealed that the RNA substrate, when prebound to I-AniI, stoichiometrically inhibits DNA cleavage activity, yet in reciprocal experiments, saturating amounts of prebound DNA substrate fails to inhibit RNA splicing activity. The data suggest therefore that both nucleic acids do not bind the same single binding site, rather that I-AniI appears to contain two binding sites.     

Degenerated recognition property of a mitochondrial homing enzyme in the unicellular green alga Chlamydomonas smithii

Target sequence cleavage is the essential step for intron invasion into an intronless allele. DNA cleavage at a specific site is performed by an endonuclease, termed a homing enzyme, which is encoded by an open reading frame within the intron. The recognition properties of them have only been analyzed in vitro, using purified, recombinant homing enzyme and various mutated DNA substrates, but it is unclear whether the homing enzyme behaves similarly in vivo. To answer this question, we determined the recognition properties of I-CsmI in vivo. I-CsmI is a homing enzyme encoded by the open reading frame of the alpha-group I-intron, located in the mitochondrial apocytochrome b gene of the green alga Chlamydomonas smithii. The in vivo recognition properties of it were determined as the frequency of intron invasion into a mutated target site. For this purpose, we utilized hybrid diploid cells developed by crossing alpha-intron-plus C. smithii to intron-minus C. reinhardtii containing mutated target sequences. The intron invasion frequency was much higher than the expected from the in vitro cleavage frequency of the respective mutated substrates. Even the substrates that had very little cleavage in the in vitro experiment were efficiently invaded in vivo, and were accompanied by a large degree of coconversion. Considering the ease of the homing enzyme invading into various mutated target sequences, we propose that the principle bottleneck for lateral intron transmission is not the sequence specificity of the homing enzyme, but instead is limited by the rare occurrence of inter-specific cell fusion.     

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.     

The I-CeuI endonuclease recognizes a sequence of 19 base pairs and preferentially cleaves the coding strand of the Chlamydomonas moewusii chloroplast large subunit rRNA gene.

The I-CeuI endonuclease is a member of the growing family of homing endonucleases that catalyse mobility of group I introns by making a double-strand break at the homing site of these introns in cognate intronless alleles during genetic crosses. In a previous study, we have shown that a short DNA fragment of 26 bp, encompassing the homing site of the fifth intron in the Chlamydomonas eugametos chloroplast large subunit rRNA gene (Ce LSU.5), was sufficient for I-CeuI recognition and cleavage. Here, we report the recognition sequence of the I-CeuI endonuclease, as determined by random mutagenesis of nucleotide positions adjacent to the I-CeuI cleavage site. Single-base substitutions that completely abolish endonuclease activity delimit a 15-bp sequence whereas those that reduce the cleavage rate define a 19-bp sequence that extends from position -7 to position +12 with respect to the Ce LSU.5 intron insertion site. As the other homing endonucleases that have been studied so far, the I-CeuI endonuclease recognizes a non-symmetric degenerate sequence. The top strand of the recognition sequence is preferred for I-CeuI cleavage and the bottom strand most likely determines the rate of double-strand breaks.     

The recent transfer of a homing endonuclease gene

The myxomycete Didymium iridis (isolate Panama 2) contains a mobile group I intron named Dir.S956-1 after position 956 in the nuclear small subunit (SSU) rRNA gene. The intron is efficiently spread through homing by the intron-encoded homing endonuclease I-DirI. Homing endonuclease genes (HEGs) usually spread with their associated introns as a unit, but infrequently also spread independent of introns (or inteins). Clear examples of HEG mobility are however sparse. Here, we provide evidence for the transfer of a HEG into a group I intron named Dir.S956-2 that is inserted into the SSU rDNA of the Costa Rica 8 isolate of D.iridis. Similarities between intron sequences that flank the HEG and rDNA sequences that flank the intron (the homing endonuclease recognition sequence) suggest that the HEG invaded the intron during the recent evolution in a homing-like event. Dir.S956-2 is inserted into the same SSU site as Dir.S956-1. Remarkably, the two group I introns encode distantly related splicing ribozymes with phylogenetically related HEGs inserted on the opposite strands of different peripheral loop regions. The HEGs are both interrupted by small spliceosomal introns that must be removed during RNA maturation.     

The structure of I-CeuI homing endonuclease: Evolving asymmetric DNA recognition from a symmetric protein scaffold

Homing endonucleases are highly specific catalysts of DNA strand breaks, leading to the transfer of mobile intervening sequences containing the endonuclease ORF. We have determined the structure and DNA recognition behavior of I-CeuI, a homodimeric LAGLIDADG endonuclease from Chlamydomonas eugametos. This symmetric endonuclease displays unique structural elaborations on its core enzyme fold, and it preferentially cleaves a highly asymmetric target site. This latter property represents an early step, prior to gene fusion, in the generation of asymmetric DNA binding platforms from homodimeric ancestors. The divergence of the sequence, structure, and target recognition behavior of homing endonucleases, as illustrated by this study, leads to the invasion of novel genomic sites by mobile introns during evolution.     

Activity and Specificity of the Bacterial PD-(D/E)XK Homing Endonuclease I-Ssp6803I

The restriction endonuclease fold [a three-layer α-β sandwich containing variations of the PD-(D/E)XK nuclease motif] has been greatly diversified during evolution, facilitating its use for many biological functions. Here we characterize DNA binding and cleavage by the PD-(D/E)XK homing endonuclease I-Ssp6803I. Unlike most restriction endonucleases harboring the same core fold, the specificity profile of this enzyme extends over a long (17 bp) target site. The DNA binding and cleavage specificity profiles of this enzyme were independently determined and found to be highly correlated. However, the DNA target sequence contains several positions where binding and cleavage activities are not tightly coupled: individual DNA base-pair substitutions at those positions that significantly decrease cleavage activity have minor effects on binding affinity. These changes in the DNA target sequence appear to correspond to substitutions that uniquely increase the free energy change between the ground state and the transition state, rather than simply decreasing the overall DNA binding affinity. The specificity of the enzyme reflects constraints on its host gene and limitations imposed by the enzyme's quaternary structure and illustrate the highly diverse repertoire of DNA recognition specificities that can be adopted by the related folds surrounding the PD-(D/E)XK nuclease motif.     

A functional homing endonuclease in the Bacillus anthracis nrdE group I intron

The essential Bacillus anthracis nrdE gene carries a self-splicing group I intron with a putative homing endonuclease belonging to the GIY-YIG family. Here, we show that the nrdE pre-mRNA is spliced and that the homing endonuclease cleaves an intronless nrdE gene 5 nucleotides (nt) upstream of the intron insertion site, producing 2-nt 3' extensions. We also show that the sequence required for efficient cleavage spans at least 4 bp upstream and 31 bp downstream of the cleaved coding strand. The position of the recognition sequence in relation to the cleavage position is as expected for a GIY-YIG homing endonuclease. Interestingly, nrdE genes from several other Bacillaceae were also susceptible to cleavage, with those of Bacillus cereus, Staphylococcus epidermidis (nrdE1), B. anthracis, and Bacillus thuringiensis serovar konkukian being better substrates than those of Bacillus subtilis, Bacillus lichenformis, and S. epidermidis (nrdE2). On the other hand, nrdE genes from Lactococcus lactis, Escherichia coli, Salmonella enterica serovar Typhimurium, and Corynebacterium ammoniagenes were not cleaved. Intervening sequences (IVSs) residing in protein-coding genes are often found in enzymes involved in DNA metabolism, and the ribonucleotide reductase nrdE gene is a frequent target for self-splicing IVSs. A comparison of nrdE genes from seven gram-positive low-G+C bacteria, two bacteriophages, and Nocardia farcinica showed five different insertion sites for self-splicing IVSs within the coding region of the nrdE gene.     

DNA substrate specificity and cleavage kinetics of an archaeal homing-type endonuclease from Pyrobaculum organotrophum.

The protein encoded by intron 1 of the single 23S rRNA gene of the archaeal hyperthermophile Pyrobaculum organotrophum was isolated and shown to constitute a homing-type DNA endonuclease, I-PorI. It cleaves the intron- 23S rDNA of the closely related organism Pyrobaculum islandicum near the site of intron insertion in Pb.organotrophum. In contrast, no endonuclease activity was detected for the protein product of intron 2 of the same gene of Pb.organotrophum which, like I-PorI, carries the LAGLI-DADG motif, common to group I intron-encoded homing enzymes. I-PorI cleaves optimally at 80 degrees C, with kcat and Km values of about 2 min-1 and 4 nM, respectively, and generates four nucleotide 3'-overhangs and 5'-phosphates. It can cleave a 25 base pair DNA fragment encompassing the intron insertion site. A mutation-selection study established the base pair specificity of the endonuclease within a 17 bp region, from positions -6 to +11 with respect to the intron-insertion site. Four of the essential base pairs encode the sequence involved in the intron-exon interaction in the pre-rRNA that is required for recognition by the RNA splicing enzymes. Properties of the enzyme are compared and contrasted with those of eucaryotic homing endonucleases.     

Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation

The LAGLIDADG homing endonuclease (LHE) I-AniI has adopted an extremely efficient secondary RNA splicing activity that is beneficial to its host, balanced against inefficient DNA cleavage. A selection experiment identified point mutations in the enzyme that act synergistically to improve endonuclease activity. The amino-acid substitutions increase target affinity, alter the thermal cleavage profile and significantly increase targeted recombination in transfected cells. The RNA splicing activity is not affected by these mutations. The improvement in DNA cleavage activity is largely focused on one of the enzyme's two active sites, corresponding to a rearrangement of a lysine residue hypothesized to act as a general base. Most of the constructs isolated in the screen contain one or more mutations that revert an amino-acid identity to a residue found in one or more close homologues of I-AniI. This implies that mutations that have previously reduced the endonuclease activity of I-AniI are identified and reversed, sometimes in combination with additional ‘artificial’ mutations, to optimize its in vivo activity.     

Structure-Function Studies of Two Yeast Homing Endonucleases that Evolved to Cleave Identical Targets with Dissimilar Rates and Specificities

The LAGLIDADG family of homing endonucleases (LHEs) bind to and cleave their DNA recognition sequences with high specificity. Much of our understanding for how these proteins evolve their specificities has come from studying LHE homologues. To gain insight into the molecular basis of LHE specificity, we characterized I-WcaI, the homologue of the Saccharomyces cerevisiae I-SceI LHE found in Wickerhamomyces canadensis. Although I-WcaI and I-SceI cleave the same recognition sequence, expression of I-WcaI, but not I-SceI, is toxic in bacteria. Toxicity suppressing mutations frequently occur at I-WcaI residues critical for activity and I-WcaI cleaves many more non-cognate sequences in the Escherichia coli genome than I-SceI, suggesting I-WcaI endonuclease activity is the basis of toxicity. In vitro, I-WcaI is a more active and a less specific endonuclease than I-SceI, again accounting for the observed toxicity in vivo. We determined the X-ray crystal structure of I-WcaI bound to its cognate target site and found that I-WcaI and I-SceI use residues at different positions to make similar base-specific contacts. Furthermore, in some regions of the DNA interface where I-WcaI specificity is lower, the protein makes fewer DNA contacts than I-SceI. Taken together, these findings demonstrate the plastic nature of LHE site recognition and suggest that I-WcaI and I-SceI are situated at different points in their evolutionary pathways towards acquiring target site specificity.