Intron-encoded Homing Endonuclease Research Articles

An intron endonuclease facilitates interference competition between coinfecting viruses.

Introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. In this work, we studied intron-encoded homing endonuclease gp210 in bacteriophage ΦPA3 and found that it contributes to viral competition by interfering with the replication of a coinfecting phage, ΦKZ. We show that gp210 targets a specific sequence in ΦKZ, which prevents the assembly of progeny viruses. This work demonstrates how a homing endonuclease can be deployed in interference competition among viruses and provide a relative fitness advantage. Given the ubiquity of homing endonucleases, this selective advantage likely has widespread evolutionary implications in diverse plasmid and viral competition as well as virus-host interactions.

Comparative Genomics Unveils Regionalized Evolution of the Faustovirus Genomes.

Faustovirus is a recently discovered genus of large DNA virus infecting the amoeba Vermamoeba vermiformis, which is phylogenetically related to Asfarviridae. To better understand the diversity and evolution of this viral group, we sequenced six novel Faustovirus strains, mined published metagenomic datasets and performed a comparative genomic analysis. Genomic sequences revealed three consistent phylogenetic groups, within which genetic diversity was moderate. The comparison of the major capsid protein (MCP) genes unveiled between 13 and 18 type-I introns that likely evolved through a still-active birth and death process mediated by intron-encoded homing endonucleases that began before the Faustovirus radiation. Genome-wide alignments indicated that despite genomes retaining high levels of gene collinearity, the central region containing the MCP gene together with the extremities of the chromosomes evolved at a faster rate due to increased indel accumulation and local rearrangements. The fluctuation of the nucleotide composition along the Faustovirus (FV) genomes is mostly imprinted by the consistent nucleotide bias of coding sequences and provided no evidence for a single DNA replication origin like in circular bacterial genomes.

Recombineering and I-SceI-mediated Pseudomonas putida KT2440 scarless gene deletion.

Pseudomonas putida KT2440 is a saprophytic and generally recognized as safe microorganism that plays important roles in the biodegradation and production of value-added chemicals. Chromosomal gene deletion of P. putida KT2440 usually involves time-consuming gene cloning, conjugal transfer and counterselection. Recently, we developed a P. putida KT2440 markerless gene deletion method based on recombineering and Cre/loxP site-specific recombination. PCR-based λ Red recombineering circumvents the tedious cloning steps and is more amenable to high-throughput manipulation. Here we report an improved scarless gene deletion strategy based on recombineering and intron-encoded homing endonuclease I-SceI-mediated double-strand break repair. Sixteen drug exporter gene(s) were deleted and the minimal inhibition concentrations of the mutants to a variety of antibiotics were determined. The robustness of the procedure was also demonstrated by sequential deletion of five large genomic regions. Up to 59% recombination efficiency was achieved for a 54.8 kb deletion, and the efficiency of RecA-mediated double-strand break repair, which was boosted by λ Red recombinase, was nearly 100%. The strain with a 3.76% genome reduction showed an improved growth rate and transformation efficiency. The straightforward, time-saving and highly efficient scarless deletion approach has the potential to facilitate the genetic study, and biotechnological and environmental applications of P. putida KT2440.

I-OmiI and I-OmiII: Two intron-encoded homing endonucleases within the Ophiostoma minus rns gene

The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus [strain WIN(M)371] was found to contain a group IC2 and a group IIB1 intron at positions mS569 and mS952 respectively. Both introns have open reading frames (ORFs) embedded that encode double motif LAGLIDADG homing endonucleases (I-OmiI and I-OmiII respectively). Codon-optimized versions of I-OmiI and I-OmiII were synthesized for overexpression in Escherichia coli. The in vitro characterization of I-OmiII showed that it is a functional homing endonuclease that cleaves the rns target site two nucleotides upstream (sense strand) of the intron insertion site generating 4 nucleotide 3′ overhangs. The endonuclease activity of I-OmiII was tested using linear and circular substrates and cleavage activity was evaluated at various temperatures. The I-OmiI protein was expressed in E. coli, but purification was difficult, thus the endonuclease activity of this protein was tested via in vivo assays. Overall this study showed that there are many native forms of functional homing endonucleases yet to be discovered among fungal mtDNA genomes.

Long non-coding genes implicated in response to stripe rust pathogen stress in wheat (Triticum aestivum L.)

The non-protein-coding genes have been reported as a critical control role in the regulation of gene expression in abiotic stress. We previously identified four expressed sequence tags numbered S18 (EL773024), S73 (EL773035), S106 (EL773041) and S108 (EL773042) from a SSH-cDNA library of bread wheat Shaanmai 139 infected with Puccinia striiformis f. sp. tritici (Pst). Here, we isolated four cDNA clones and referred them as TalncRNA18, TalncRNA73, TalncRNA106 and TalncRNA108 (GenBank: KC549675-KC549678). These cDNA separately consisted of 1,393, 667, 449 and 647 nucleotides but without any open reading frame. The alignment result showed that TalncRNA18 is a partial cDNA of E3 ubiquitin-protein ligase UPL1-like gene, TalncRNA73 is an antisense transcript of hypothetical protein, TalncRNA108 is a homolog to RRNA intron-encoded homing endonuclease, and lncRNA106 had no similarly sequence. Quantitative RT-PCR studies confirmed that these four lncRNAs were differentially expressed in three near isogenic lines. TalncRNA108 was significantly stepwise decreased at early stage of inoculation with Pst, while the others were upregulated, especially at 1 and 3 dpi (days post-inoculation). Using Chinese Spring nulli-tetrasomic lines and its ditelosomic lines, TalncRNA73 and TalncRNA108 were located to wheat chromosome 7A and the short arm of chromosome 4B, respectively, while TalncRNA18 and TalncRNA106 were located to chromosome 5B. Comparing the sequence of DNA and cDNA of four lncRNAs with polymerase chain reaction primers, the results showed that all of them have no introns. The kinetics analyses of lncRNAs expression as a result of pathogen challenge in immune resistant genotype indicated that they may play the roles of modulating or silencing the protein-coding gene into pathogen-defence response.

Invasion of protein coding genes by green algal ribosomal group I introns

The spread of group I introns depends on their association with intron-encoded homing endonucleases. Introns that encode functional homing endonuclease genes (HEGs) are highly invasive, whereas introns that only encode the group I ribozyme responsible for self-splicing are generally stably inherited (i.e., vertical inheritance). A number of recent case studies have provided new knowledge on the evolution of group I introns, however, there are still large gaps in understanding of their distribution on the tree of life, and how they have spread into new hosts and genic sites. During a larger phylogenetic survey of chlorophyceaen green algae, we found that 23 isolates contain at least one group I intron in the rbcL chloroplast gene. Structural analyses show that the introns belong to one of two intron lineages, group IA2 intron-HEG (GIY-YIG family) elements inserted after position 462 in the rbcL gene, and group IA1 introns inserted after position 699. The latter intron type sometimes encodes HNH homing endonucleases. The distribution of introns was analyzed on an exon phylogeny and patterns were recovered that are consistent with vertical inheritance and possible horizontal transfer. The rbcL 462 introns are thus far reported only within the Volvocales, Hydrodictyaceae and Bracteacoccus, and closely related isolates of algae differ in the presence of rbcL introns. Phylogenetic analysis of the intron conserved regions indicates that the rbcL699 and rbcL462 introns have distinct evolutionary origins. The rbcL699 introns were likely derived from ribosomal RNA L2449 introns, whereas the rbcL462 introns form a close relationship with psbA introns.

Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element

Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)2 large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.

Evolution of Pleopsidium (Lichenized Ascomycota) S943 Group I Introns and the Phylogeography of an Intron-Encoded Putative Homing Endonuclease

The sporadic distribution of nuclear group I introns among different fungal lineages can be explained by vertical inheritance of the introns followed by successive losses, or horizontal transfers from one lineage to another through intron homing or reverse splicing. Homing is mediated by an intron-encoded homing endonuclease (HE) and recent studies suggest that the introns and their associated HE gene (HEG) follow a recurrent cyclical model of invasion, degeneration, loss, and reinvasion. The purpose of this study was to compare this model to the evolution of HEGs found in the group I intron at position S943 of the nuclear ribosomal DNA of the lichen-forming fungus Pleopsidium. Forty-eight S943 introns were found in the 64 Pleopsidium samples from a worldwide screen, 22 of which contained a full-length HEG that encodes a putative 256-amino acid HE, and 2 contained HE pseudogenes. The HEGs are divided into two closely related types (as are the introns that encode them) that differ by 22.6% in their nucleotide sequences. The evolution of the Pleopsidium intron-HEG element shows strong evidence for a cyclical model of evolution. The intron was likely acquired twice in the genus and then transmitted via two or three interspecific horizontal transfers. Close geographical proximity plays an important role in intron-HEG horizontal transfer because most of these mobile elements were found in Europe. Once acquired in a lineage, the intron-HEG element was also vertically transmitted, and occasionally degenerated or was lost.

Computational redesign of endonuclease DNA binding and cleavage specificity

The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site approximately 10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages

T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes.

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.

Intron-encoded homing endonuclease I-TevI also functions as a transcriptional autorepressor.

Customary binding sites of intron-encoded homing endonucleases lie within cognate intronless alleles, at the so-called homing sites. Here, we describe a novel, high-affinity binding site for I-TevI endonuclease, encoded within the group I td intron of phage T4. This site is an operator that overlaps the T4 late promoter, which drives I-TevI expression from within the td intron. I-TevI binds the operator and homing sites with equal affinity, and functions as a transcriptional autorepressor. Distinct sequence and spacing requirements of the catalytic domain result in reduced cleavage activity on operator DNA. Crystallographic studies showed that the overall interactions of the DNA-binding domain with the operator and homing sites are similar, but have some different hydrogen-bonding contacts. We present a model in which the flexibility in protein-DNA interactions allows I-TevI to bind variant intronless alleles to promote intron mobility while facilitating its function in autorepression, and thereby persistence in its host.

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.

Characterization of the I-Spom I endonuclease from fission yeast: insights into the evolution of a group I intron-encoded homing endonuclease.

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli. Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo, the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro. Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.

Structural and mechanistic basis of immunity toward endonuclease colicins.

The crystal structure of the cytotoxic endonuclease domain from the bacterial toxin colicin E9 in complex with its cognate immunity protein Im9 reveals that the inhibitor does not bind at the active site, the core of which comprises the HNH motif found in intron-encoded homing endonucleases, but rather at an adjacent position leaving the active site exposed yet unable to bind DNA because of steric and electrostatic clashes with incoming substrate. Although its mode of action is unorthodox, Im9 is a remarkably effective inhibitor since it folds within milliseconds and then associates with its target endonuclease at the rate of diffusion to form an inactive complex with sub-femtomolar binding affinity. This hyperefficient mechanism of inhibition could be well suited to other toxic enzyme systems, particularly where the substrate is a polymer extending beyond the boundaries of the active site.

The structure, function, and convergent evolution of intron-encoded homing endonucleases

The structure, function, and convergent evolution of intron-encoded homing endonucleases

DNA Recognition and Cleavage by the LAGLIDADG Homing Endonuclease I- Cre I

The structure of the LAGLIDADG intron-encoded homing endonuclease I- Cre I bound to homing site DNA has been determined. The interface is formed by an extended, concave β sheet from each enzyme monomer that contacts each DNA half-site, resulting in direct side-chain contacts to 18 of the 24 base pairs across the full-length homing site. The structure indicates that I- Cre I is optimized to its role in genetic transposition by exhibiting long site-recognition while being able to cleave many closely related target sequences. DNA cleavage is mediated by a compact pair of active sites in the I- Cre I homodimer, each of which contains a separate bound divalent cation.

DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI.

Homing endonucleases are a diverse collection of proteins that are encoded by genes with mobile, self-splicing introns. They have also been identified in self-splicing inteins (protein introns). These enzymes promote the movement of the DNA sequences that encode them from one chromosome location to another; they do this by making a site-specific double-strand break at a target site in an allele that lacks the corresponding mobile intron. The target sites recognized by these small endonucleases are generally long (14-44 base pairs). Four families of homing endonucleases have been identified, including the LAGLIDADG, the His-Cys box, the GIY-YIG and the H-N-H endonucleases. The first identified His-Cys box homing endonuclease was I-PpoI from the slime mould Physarum polycephalum. Its gene resides in one of only a few nuclear introns known to exhibit genetic mobility. Here we report the structure of the I-PpoI homing endonuclease bound to homing-site DNA determined to 1.8 A resolution. I-PpoI displays an elongated fold of dimensions 25 x 35 x 80 A, with mixed alpha/beta topology. Each I-PpoI monomer contains three antiparallel beta-sheets flanked by two long alpha-helices and a long carboxy-terminal tail, and is stabilized by two bound zinc ions 15 A apart. The enzyme possesses a new zinc-bound fold and endonuclease active site. The structure has been determined in both uncleaved substrate and cleaved product complexes.

Mapping metal ions at the catalytic centres of two intron-encoded endonucleases.

Divalent metal ions play a crucial role in forming the catalytic centres of DNA endonucleases. Substitution of Mg2+ ions by Fe2+ ions in two archaeal intron-encoded homing endonucleases, I-DmoI and I-PorI, yielded functional enzymes and enabled the generation of reactive hydroxyl radicals within the metal ion binding sites. Specific hydroxyl radical-induced cleavage was observed within, and immediately after, two conserved LAGLIDADG motifs in both proteins and at sites at, and near, the scissile phosphates of the corresponding DNA substrates. Titration of Fe2+-containing protein-DNA complexes with Ca2+ ions, which are unable to support endonucleolytic activity, was performed to distinguish between the individual metal ions in the complex. Mutations of single amino acids in this region impaired catalytic activity and caused the preferential loss of a subset of hydroxyl radical cleavages in both the protein and the DNA substrate, suggesting an active role in metal ion coordination for these amino acids. The data indicate that the endonucleases cleave their DNA substrates as monomeric enzymes, and contain a minimum of four divalent metal ions located at or near the catalytic centres of each endonuclease. The metal ions involved in cleaving the coding and the non-coding strand are positioned immediately after the N- and C-terminally located LAGLIDADG motifs, respectively. The dual protein/nucleic acid footprinting approach described here is generally applicable to other protein-nucleic acid complexes when the natural metal ion can be replaced by Fe2+.