Homing Endonuclease Domain Research Articles

Structural Basis for the Propagation of Homing Endonuclease-Associated Inteins.

Inteins catalyze their removal from a host protein through protein splicing. Inteins that contain an additional site-specific endonuclease domain display genetic mobility via a process termed “homing” and thereby act as selfish DNA elements. We elucidated the crystal structures of two archaeal inteins associated with an active or inactive homing endonuclease domain. This analysis illustrated structural diversity in the accessory domains (ACDs) associated with the homing endonuclease domain. To augment homing endonucleases with highly specific DNA cleaving activity using the intein scaffold, we engineered the ACDs and characterized their homing site recognition. Protein engineering of the ACDs in the inteins illuminated a possible strategy for how inteins could avoid their extinction but spread via the acquisition of a diverse accessory domain.

An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein

Protein splicing is a post-translational process by which an intein catalyzes its own excision from flanking polypeptides, or exteins, concomitant with extein ligation. Many inteins have nested homing endonuclease domains that facilitate their propagation into intein-less alleles, whereas other inteins lack the homing endonuclease (HEN) and are called mini-inteins. The mini-intein that interrupts the DNA PolII of Pyrococcus horikoshii has a linker region in place of the HEN domain that is shorter than the linker in a closely related intein from Pyrococcus abyssi. The P. horikoshii PolII intein requires a higher temperature for catalytic activity and is more stable to digestion by the thermostable protease thermolysin, suggesting that it is more rigid than the P. abyssi intein. We solved a crystal structure of the intein precursor that revealed a domain-swapped dimer. Inteins found as domain swapped dimers have been shown to promote intein-mediated protein alternative splicing, but the solved P. horikoshii PolII intein structure has an active site unlikely to be catalytically competent.

Inteins in Science: Evolution to Application.

Inteins are mobile genetic elements that apply standard enzymatic strategies to excise themselves post-translationally from the precursor protein via protein splicing. Since their discovery in the 1990s, recent advances in intein technology allow for them to be implemented as a modern biotechnological contrivance. Radical improvement in the structure and catalytic framework of cis- and trans-splicing inteins devised the development of engineered inteins that contribute to various efficient downstream techniques. Previous literature indicates that implementation of intein-mediated splicing has been extended to in vivo systems. Besides, the homing endonuclease domain also acts as a versatile biotechnological tool involving genetic manipulation and control of monogenic diseases. This review orients the understanding of inteins by sequentially studying the distribution and evolution pattern of intein, thereby highlighting a role in genetic mobility. Further, we include an in-depth summary of specific applications branching from protein purification using self-cleaving tags to protein modification, post-translational processing and labelling, followed by the development of intein-based biosensors. These engineered inteins offer a disruptive approach towards research avenues like biomaterial construction, metabolic engineering and synthetic biology. Therefore, this linear perspective allows for a more comprehensive understanding of intein function and its diverse applications.

Protein Splicing Activity of the Haloferax volcanii PolB-c Intein Is Sensitive to Homing Endonuclease Domain Mutations.

Inteins are selfish genetic elements residing in open reading frames that can splice post-translationally, resulting in the ligation of an uninterrupted, functional protein. Like other inteins, the DNA polymerase B (PolB) intein of the halophilic archaeon Haloferax volcanii has an active homing endonuclease (HEN) domain, facilitating its horizontal transmission. Previous work has shown that the presence of the PolB intein exerts a significant fitness cost on the organism compared to an intein-free isogenic H. volcanii. Here, we show that mutation of a conserved residue in the HEN domain not only reduces intein homing but also slows growth. Surprisingly, although this mutation is far from the protein splicing active site, it also significantly reduces in vitro protein splicing. Moreover, two additional HEN domain mutations, which could not be introduced to H. volcanii, presumably due to lethality, also eliminate protein splicing activity in vitro. These results suggest an interplay between HEN residues and the protein splicing domain, despite an over 35 Å separation in a PolB intein homology model. The combination of in vivo and in vitro evidence strongly supports a model of codependence between the self-splicing domain and the HEN domain that has been alluded to by previous in vitro studies of protein splicing with HEN domain-containing inteins.

Homing Endonuclease and Protein Splicing Activity of Inteins from Thermophilic Archaea

Protein splicing is a post translational, self‐catalyzed reaction by which an intein removes itself from flanking polypeptides and ligates those polypeptides together. We are interested in the splicing and homing endonuclease activities of the intein that interrupt the DNA polymerase II in the extreme thermophiles Thermococcus barophilus and Thermococcus kodakarensis. Given that T. kodakarensis is a surface‐dwelling organism whereas the T. barophilus is a piezophile that lives in deep‐sea thermal vents, yet both inteins are highly similar in sequence, we are interested in how temperature and pressure may affect enzyme activity. Additionally, we are interested in the role of the two conserved C‐terminal residues of the intein. We have found that the homing endonuclease domain of the T. kodakarensis intein is more active at a lower concentration and lower temperatures than that of the T. barophilus intein. Protein splicing of both inteins can be induced by in vitro incubation of an isolated, unspliced precursor at elevated temperature, which may also be influenced by hydrostatic pressure.Support or Funding InformationThis work was supported by NSF grant MCB‐1517138, a Henry Dreyfus Teacher‐Scholar Award, and by NIH Grant 1R15GM132817‐0

Protein splicing and tryptophan fluorescence of salt‐dependent inteins from Haloquadratum walsbyi

Protein splicing is a self‐catalyzed reaction by which an intein, an intervening polypeptide, catalyzes its own removal from exteins, the flanking polypeptides, as well as ligation of the exteins. We are studying the inteins that interrupt a cell division control protein in the halophilic archaea Haloquadratum walsbyi. Halophiles like H. walsbyi are able to survive in water with high concentrations of sodium chloride. H. walsbyi has four inteins that interrupt the cdc21 gene, and we have studied the two inteins (Hwa cdc21A and Hwa cdc21D) that lack an intervening homing endonuclease domain. We are studying the splicing activity under various salt concentrations for the inteins both separately and when expressed as part of a single fusion protein. Hwa cdc21A splices optimally at 2.5 M NaCl, while Hwa cdc21D splices optimally at 1.5 M NaCl. We also are using native Trp fluorescence to study the influence of salt concentration on the proper folding of the inteins.Support or Funding InformationThis work was supported by NSF grant MCB‐1517138, a Henry Dreyfus Teacher‐Scholar Award, and NIH Grant 1R15GM132817‐01.

Salt‐dependent protein splicing of an intein from the halophile Haloferax volcanii

Protein splicing is a post‐translational process facilitated by an intervening polypeptide called an intein, or internal protein. The intein is located between two flanking polypeptides, called exteins. The intein catalyzes its excision from the extein concomitant with the ligation of the exteins. The extremophile Haloferax volcanii grows in high salt environments. We are interested in the salt‐dependence of the protein splicing and nuclease activity of the intein that has invaded its DNA Polymerase beta gene. We have shown that we can induce protein splicing of the intein in vitro as a function of salt concentration and time. The intein has an intervening homing endonuclease domain and we are interested to learn if mutations within this domain would influence splicing. Characterization of a conditionally‐active nuclease may have utility in biotechnology applications.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138), National Institutes of Health (grant 1R15GM132817‐01), and by the Camille & Henry Dreyfus Foundation

Crystal structures of CDC21-1 inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site

Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly conserved insertion site. A purifying selection mechanism directing the location of the HEN insertion site has not yet been identified. In this work, we solved the three-dimensional crystal structures of two inteins inserted in the cell division control protein 21 of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii. A comparison between the structures provides the structural basis for the thermo-stabilization mechanism of inteins that have lost the HEN domain during evolution. The presence of an entire extein domain in the intein structure from Pyrococcus horikoshii suggests the selection mechanism for the highly conserved HEN insertion point.

PRP8 Intein in Onygenales: Distribution and Phylogenetic Aspects.

Inteins (internal proteins) are mobile genetic elements, inserted in housekeeping proteins, with self-splicing properties. Some of these elements have been recently pointed out as modulators of genetic expression or protein function. Herein, we evaluated, in silico, the distribution and phylogenetic patterns of PRP8 intein among 93 fungal strains of the order Onygenales. PRP8 intein(s) are present in most of the species (45/49), mainly as full-length inteins (containing both the Splicing and the Homing Endonuclease domains), and must have transferred vertically in all lineages, since their phylogeny reflects the group phylogeny. While the distribution of PRP8 intein(s) varies among species of Onygenaceae family, being absent in Coccidioides spp. and present as full and mini-intein in other species, they are consistently observed as full-length inteins in all evaluated pathogenic species of the Arthrodermataceae and Ajellomycetaceae families. This conservative and massive PRP8 intein presence in Ajellomycetacean and Arthrodermatecean species reinforces the previous idea that such genetic elements do not decrease the fungal fitness significantly and even might play some role in the host-pathogen relationship, at least in these two fungal groups. We may better position the species Ophidiomyces ophiodiicola (with no intein) in the Onygenaceae family and Onygena corvina (with a full-length intein) as a basal member in the Arthrodermataceae family.

Conditional Protein Splicing of Inteins from Extremophiles

Protein splicing is a post‐translational process facilitated by an intein, by which the intein catalyzes its own excision from flanking polypeptides (the exteins), concomitant with ligation of the exteins. Many inteins are themselves interrupted by homing endonuclease domains that can facilitate homing into intein‐less alleles. We have discovered inteins from extremophiles that can be expressed in E. coli, purified as unspliced precursors, and induced to splice by cues such as incubation under reducing conditions, at high temperature, or at high ionic strength. We have shown that the activity and folding of inteins from the extreme halophiles Halobacterium salinarum and Haloquadratum walsbyi demonstrate variable dependence on salt concentration, and we have designed transgenic organisms lacking inteins to test the physiological role of splicing. We have solved the structure of an intein from the extreme thermophile Pyrococcus abyssi, and studied the influence of structural elements on its stability at high temperature and pressure. We also have discovered that the splicing and nuclease activity of inteins from extreme thermophiles can be influenced by pressures consistent with those found at the seafloor.Support or Funding InformationThis work was supported by grants from the National Science Foundation (grants MCB1244089 and MCB1517138) and the Camille and Henry Dreyfus Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Salt dependence of a self‐splicing enzyme from an extreme halophile

Protein splicing is a post‐translational process facilitated by an intervening polypeptide called an internal protein, or intein. The intein is located between two flanking polypeptides, called exteins. The intein catalyzes its excision from the extein concomitantly with the ligation of the exteins. The extremophile Haloferax volcanii exists in a high salt environment. We are interested in the halophile because we want to learn how efficiently the intein that has invaded its PolB gene splices as a function of salt concentration. We have shown that we can induce splicing of the intein in vitro as a function of salt concentration and time. Others (http://www.ncbi.nlm.nih.gov/pubmed/27462108) have demonstrated that the intein has an active homing endonuclease domain in vivo, and that the presence of the intein has a high fitness cost for the organism. We are interested in studying the endonuclease activity of the intein in vitro, given that a salt‐dependent conditionally‐active nuclease may have utility in biotechnology applications.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

“The relationship of structural stability to temperature‐dependent activity in a family of thermophilic inteins”

Protein splicing is a post‐translational process by which a self‐catalyzing intervening sequence called an intein removes itself from a precursor protein and concomitantly ligates the flanking sequences, known as exteins. Our inteins of interest interrupt the DNA Polymerase II from the extreme thermophiles Thermococcus sibiricus (Tsi) and Thermococcus gammatolerans (Tga). We are interested in these inteins because of their sequence similarity to those from the extreme thermophiles Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho), all of which are mini‐inteins that lack a homing endonuclease domain. The Pho intein has been found to require higher temperature for splicing than the Pab intein, perhaps due to a shorter, more conformationally‐restricted region that replaces the missing homing endonuclease domain. The Tga and Tsi inteins have loop lengths more similar to the Pho intein. A splicing assay under increasing pressure will determine if there is a difference in activity in the two inteins that vary in the depths at which the parent organisms dwell. A preliminary assay at atmospheric pressure with an optimal isothermal temperature of 52°C showed that the Tga intein splices more efficiently than the Tsi intein. We will measure the temperature dependence of the intrinsic tryptophan fluorescence to link the overall stability of the fold to the difference in activity.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Camille and Henry Dreyfus Foundation (KVM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Homing Endonuclease and Protein Splicing Activity of Inteins from Extreme Thermophiles

Protein splicing is a post‐translational modification by which intervening polypeptides, or inteins, catalyze their own excision from two flanking polypeptides, or exteins, concomitant with the ligation of the exteins. Many inteins contain a homing endonuclease domain, which can facilitate intein homing by catalyzing double strand breaks in intein‐less extein alleles. We studied the in vitro protein splicing and endonuclease activity of homing endonuclease‐containing inteins that interrupt the DNA Polymerase II (PolII) from the deep‐sea thermophiles Thermococcus kodakarensis (Tko) and Thermococcus barophilus (Tba). It was found that the Tba and Tko inteins can be purified as unspliced precursors and induced to splice in vitro at temperatures greater than 37°C. Additionally, when incubated at pressures up to 1500atm, the Tba and Tko inteins continue to exhibit splicing activity. Tba and Tko inteins also have temperature‐dependent endonuclease activity; the Tba intein is enzymatically active at pressures up to 1000atm. Future work will focus on identifying the active site residues of the homing endonuclease within the Tba and Tko inteins as well as determining the target sequence specificity of the enzyme. We also will determine if there is a difference in homing endonuclease activity between excised inteins and unsplicing intein precursors.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endonuclease and splicing activity of a Haloferax volcanii intein

Protein splicing is a post‐translational process facilitated by an intervening polypeptide called an internal protein, or intein. The intein is located between two flanking polypeptides, called exteins. The intein catalyzes its excision from the extein concomitantly with the ligation of the exteins. The extremophile Haloferax volcanii exists in a high salt environment. We are interested in the halophile because we want to learn how efficiently the intein that has invaded its PolB gene splices as a function of salt concentration. We have shown that we can induce splicing of the intein in vitro at high salt. Collaborators have demonstrated that the intein has an active homing endonuclease domain in vivo, and that the presence of the intein has a high fitness cost for the organism. We are interested in studying the endonuclease activity of the intein in vitro, given that a salt‐dependent conditionally‐active nuclease may have utility in biotechnology applications.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural Basis for the Persistence of Homing Endonucleases in Transcription Factor IIB Inteins

Inteins are mobile genetic elements that are spliced out of proteins after translation. Some inteins contain a homing endonuclease (HEN) responsible for their propagation. Hedgehog/INTein (HINT) domains catalyzing protein splicing and their nested HEN domains are thought to be functionally independent because of the existence of functional mini-inteins without HEN domains. Despite the lack of obvious mutualism between HEN and HINT domains, HEN domains are persistently found at one specific site in inteins, indicating their potential functional role in protein splicing. Here we report crystal structures of inactive and active mini-inteins derived from inteins residing in the transcription factor IIB of Methanococcus jannaschii and Methanocaldococcus vulcanius, revealing a novel modified HINT fold that might provide new insights into the mutualism between the HEN and HINT domains. We propose an evolutionary model of inteins and a functional role of HEN domains in inteins.

The mitochondrial genome of the plant-pathogenic fungus Stemphylium lycopersici uncovers a dynamic structure due to repetitive and mobile elements.

Stemphylium lycopersici (Pleosporales) is a plant-pathogenic fungus that has been associated with a broad range of plant-hosts worldwide. It is one of the causative agents of gray leaf spot disease in tomato and pepper. The aim of this work was to characterize the mitochondrial genome of S. lycopersici CIDEFI-216, to use it to trace taxonomic relationships with other fungal taxa and to get insights into the evolutionary history of this phytopathogen. The complete mitochondrial genome was assembled into a circular double-stranded DNA molecule of 75,911 bp that harbors a set of 37 protein-coding genes, 2 rRNA genes (rns and rnl) and 28 tRNA genes, which are transcribed from both sense and antisense strands. Remarkably, its gene repertoire lacks both atp8 and atp9, contains a free-standing gene for the ribosomal protein S3 (rps3) and includes 13 genes with homing endonuclease domains that are mostly located within its 15 group I introns. Strikingly, subunits 1 and 2 of cytochrome oxidase are encoded by a single continuous open reading frame (ORF). A comparative mitogenomic analysis revealed the large extent of structural rearrangements among representatives of Pleosporales, showing the plasticity of their mitochondrial genomes. Finally, an exhaustive phylogenetic analysis of the subphylum Pezizomycotina based on mitochondrial data reconstructed their relationships in concordance with several studies based on nuclear data. This is the first report of a mitochondrial genome belonging to a representative of the family Pleosporaceae.

Impact of a homing intein on recombination frequency and organismal fitness

Inteins are parasitic genetic elements that excise themselves at the protein level by self-splicing, allowing the formation of functional, nondisrupted proteins. Many inteins contain a homing endonuclease (HEN) domain and rely on its activity for horizontal propagation. However, successful invasion of an entire population will make this activity redundant, and the HEN domain is expected to degenerate quickly under these conditions. Several theories have been proposed for the continued existence of the both active HEN and noninvaded alleles within a population. However, to date, these models were not directly tested experimentally. Using the natural cell fusion ability of the halophilic archaeon Haloferax volcanii we were able to examine this question in vivo, by mating polB intein-positive [insertion site c in the gene encoding DNA polymerase B (polB-c)] and intein-negative cells and examining the dispersal efficiency of this intein in a natural, polyploid population. Through competition between otherwise isogenic intein-positive and intein-negative strains we determined a surprisingly high fitness cost of over 7% for the polB-c intein. Our laboratory culture experiments and samples taken from Israel's Mediterranean coastline show that the polB-c inteins do not efficiently take over an inteinless population through mating, even under ideal conditions. The presence of the HEN/intein promoted recombination when intein-positive and intein-negative cells were mated. Increased recombination due to HEN activity contributes not only to intein dissemination but also to variation at the population level because recombination tracts during repair extend substantially from the homing site.

Influence of Intein Flexibility on Temperature Dependence of Protein Splicing

Protein splicing is a post‐translational modification by which intervening polypeptides called inteins catalyze their own excision from two flanking polypeptides, the exteins, concomitant with the ligation of the exteins. We studied the activity of DNA Polymerase II (PolII) inteins from the deep‐sea thermophiles Thermococcus kodakarensis (Tko), Thermococcus barophilus (Tba), Thermococcus sibiricus (Tsib), and Methanococcus infernus (Minf). Tko and Tba PolII inteins contain a homing endonuclease domain, while a flexible loop replaces this region in the Tsib and Minf inteins. This flexible loop, also found in Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho) PolII inteins, was hypothesized to be linked to the temperature dependent activity of these inteins. However, unlike Pab and Pho PolII inteins, we have shown that protein splicing and cleavage side reactions in the Tsib and Minf inteins occur in vivo in E. coli without the need for high temperature incubation in vitro. On the other hand, the Tba and Tko inteins splice poorly in E. coli and can be induced to splice in vitro at higher temperatures. We also have demonstrated that the Tba intein splices while the Tko intein undergoes a mixture of splicing and N‐terminal cleavage.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under grants MCB‐0950245, MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).

Dynamics differentiate between active and inactive inteins.

The balance between stability and dynamics for active enzymes can be somewhat quantified by studies of intein splicing and cleaving reactions. Inteins catalyze the ligation of flanking host exteins while excising themselves. The potential for applications led to engineering of a mini-intein splicing domain, where the homing endonuclease domain of the Mycobacterium tuberculosis RecA (Mtu recA) intein was removed. The remaining domains were linked by several short peptides, but splicing activity in all was substantially lower than the full-length intein. Native splicing activity was restored in some cases by a V67L mutation. Using computations and experiments, we examine the impact of this mutation on the stability and conformational dynamics of the mini-intein splicing domain. Molecular dynamics simulations were used to delineate the factors that determine the active state, including the V67L mini-intein mutant, and peptide linker. We found that (1) the V67L mutation lowers the global fluctuations in all modeled mini-inteins, stabilizing the mini-intein constructs; (2) the connecting linker length affects intein dynamics; and (3) the flexibilities of the linker and intein core are higher in the active structure. We have observed that the interaction of the linker region and a turn region around residues 35-41 provides the pathway for the allostery interaction. Our experiments reveal that intein catalysis is characterized by non-linear Arrhenius plot, confirming the significant contribution of protein conformational dynamics to intein function. We conclude that while the V67L mutation stabilizes the global structure, cooperative dynamics of all intein regions appear more important for intein function than high stability. Our studies suggest that effectively quenching the conformational dynamics of an intein through engineered allosteric interactions could deactivate intein splicing or cleaving.

Inteins as indicators of gene flow in the halobacteria.

This research uses inteins, a type of mobile genetic element, to infer patterns of gene transfer within the Halobacteria. We surveyed 118 genomes representing 26 genera of Halobacteria for intein sequences. We then used the presence-absence profile, sequence similarity and phylogenies from the inteins recovered to explore how intein distribution can provide insight on the dynamics of gene flow between closely related and divergent organisms. We identified 24 proteins in the Halobacteria that have been invaded by inteins at some point in their evolutionary history, including two proteins not previously reported to contain an intein. Furthermore, the size of an intein is used as a heuristic for the phase of the intein's life cycle. Larger size inteins are assumed to be the canonical two domain inteins, consisting of self-splicing and homing endonuclease domains (HEN); smaller sizes are assumed to have lost the HEN domain. For many halobacterial groups the consensus phylogenetic signal derived from intein sequences is compatible with vertical inheritance or with a strong gene transfer bias creating these clusters. Regardless, the coexistence of intein-free and intein-containing alleles reveal ongoing transfer and loss of inteins within these groups. Inteins were frequently shared with other Euryarchaeota and among the Bacteria, with members of the Cyanobacteria (Cyanothece, Anabaena), Bacteriodetes (Salinibacter), Betaproteobacteria (Delftia, Acidovorax), Firmicutes (Halanaerobium), Actinobacteria (Longispora), and Deinococcus-Thermus-group.