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.     

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.     

Evolutionary dynamics of introns and their open reading frames in the U7 region of the mitochondrial rnl gene in species of Ceratocystis

The mtDNA rnl-U7 region has been examined for the presence of introns in selected species of the genus Ceratocystis. Comparative sequence analysis identified group I and group II introns encoding single and double motif LAGLIDADG open reading frames (ORFs) at the following positions L1671, L1787, and L1923. In addition downstream of the rnl-U7 region group I introns were detected at positions L1971 and L2231, and a group II intron at L2059. A GIY-YIG type ORF was located within one mL1923 LAGLIDADG type ORF and a degenerated GIY-YIG ORF fused to a nad2 gene fragment was found in association with the mL1971 group I intron. The diversity of composite elements that appear to be sporadically distributed among closely related species of Ceratocystis illustrates the potential for homing endonucleases and their associated introns to invade new sites. Phylogenetic analysis showed that single motif LADGLIDADG ORFs related to the mL1923 ORFs have invaded the L1787 group II intron and the L1671 group I intron. Phylogenetic analysis of intron encoded single and double motif LAGLIDADG ORFs also showed that these ORFs transferred four times from group I into group II B1 type introns.     

Intron open reading frames as mobile elements and evolution of a group I intron

Group I introns are proposed to have become mobile following the acquisition of open reading frames (ORFs) that encode highly specific DNA endonucleases. This proposal implies that intron ORFs could behave as autonomously mobile entities. This was supported by abundant circumstantial evidence but no experiment of ORF transfer from an ORF- containing intron to its ORF-less counterpart has been described. In this paper we present such experiments, which demonstrate the efficient mobility of the mitochondrial nad1-i4-orf1 between two Podospora strains. The homing of this mobile ORF was accompanied by a bidirectional co-conversion that did not systematically involve the whole intron sequence. Orf1 acquisition would be the most recent step in the evolution of the nad1-i4 intron, which has resulted in many strains of Podospora having an intron with two ORFs (biorfic) and four splicing pathways. We show that two of the splicing events that operate in this biorfic intron, as evidenced by PCR experiments, are generated by a 5'-alternative splice site, which is most probably a remnant of the monoorfic ancestral form of the intron. We propose a sequential evolution model that is consistent with the four organizations of the corresponding nad1 locus that we found among various species of the Pyrenomycete family; these organizations consist of no intron, an intron alone, a monoorfic intron, and a biorfic intron.      

Intron mobility in phage T4 is dependent upon a distinctive class of endonucleases and independent of DNA sequences encoding the intron core: mechanistic and evolutionary implications

Although mobility of the phylogenetically widespread group I introns appears to be mechanistically similar, the phage T4 intron-encoded endonucleases that promote mobility of the td and sunY introns are different from their eukaryotic counterparts. Most notably, they cleave at a distance from the intron insertion sites. The td enzyme was shown to cleave 23-26 nt 5' and the sunY endonuclease 13-15 nt 3' to the intron insertion site to generate 3-nt or 2-nt 3'-OH extensions, respectively. The absolute coconversion of exon markers between the distant cleavage and insertion sites is consistent with the double-strand-break repair model for intron mobility. As a further critical test of the model we have demonstrated that the mobility event is independent of DNA sequences that encode the catalytic intron core structure. Thus, in derivatives in which the lacZ or kanR coding sequences replace the intron, these marker genes are efficiently inserted into intron-minus alleles when the cognate endonuclease is provided in trans. The process is therefore endonuclease-dependent, rather than dependent on the intron per se. These findings, which imply that the endonucleases rather than the introns themselves were the primordial mobile elements, are incorporated into a model for the evolution of mobile introns.     

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.     

The nicking homing endonuclease I-BasI is encoded by a group I intron in the DNA polymerase gene of the Bacillus thuringiensis phage Bastille

Here we describe the discovery of a group I intron in the DNA polymerase gene of Bacillus thuringiensis phage Bastille. Although the intron insertion site is identical to that of the Bacillus subtilis phages SPO1 and SP82 introns, the Bastille intron differs from them substantially in primary and secondary structure. Like the SPO1 and SP82 introns, the Bastille intron encodes a nicking DNA endonuclease of the H-N-H family, I-BasI, with a cleavage site identical to that of the SPO1-encoded enzyme I-HmuI. Unlike I-HmuI, which nicks both intron-minus and intron-plus DNA, I-BasI cleaves only intron-minus alleles, which is a characteristic of typical homing endonucleases. Interestingly, the C-terminal portions of these H-N-H phage endonucleases contain a conserved sequence motif, the intron-encoded endonuclease repeat motif (IENR1) that also has been found in endonucleases of the GIY-YIG family, and which likely comprises a small DNA-binding module with a globular ββααβ fold, suggestive of module shuffling between different homing endonuclease families.     

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.     

Homing endonuclease structure and function

Homing endonucleases are encoded by open reading frames that are embedded within group I, group II and archael introns, as well as inteins (intervening sequences that are spliced and excised post-translationally). These enzymes initiate transfer of those elements (and themselves) by generating strand breaks in cognate alleles that lack the intervening sequence, as well as in additional ectopic sites that broaden the range of intron and intein mobility. Homing endonucleases can be divided into several unique families that are remarkable in several respects: they display extremely high DNA-binding specificities which arise from long DNA target sites (14-40 bp), they are tolerant of a variety of sequence variations in these sites, and they display disparate DNA cleavage mechanisms. A significant number of homing endonucleases also act as maturases (highly specific cofactors for the RNA splicing reactions of their cognate introns). Of the known homing group I endonuclease families, two (HNH and His-Cys box enzymes) appear to be diverged from a common ancestral nuclease. While crystal structures of several representatives of the LAGLIDADG endonuclease family have been determined, only structures of single members of the HNH (I-HmuI), His-Cys box (I-PpoI) and GIY-YIG (I-TevI) families have been elucidated. These studies provide an important source of information for structure-function relationships in those families, and are the centerpiece of this review. Finally, homing endonucleases are significant targets for redesign and selection experiments, in hopes of generating novel DNA binding and cutting reagents for a variety of genomic applications.     

Structural and functional characteristics of homing endonucleases

Mobile genetic elements constitute a remarkably diverse group of nonessential selfish genes that provide no apparent function to the host. These selfish genes have been implicated in host extinction, speciation and architecture of genetic systems. Homing endonucleases, encoded by the open reading frames embedded in introns or inteins of mobile genetic elements, possess double-stranded DNA-specific endonuclease activity. They inflict sequence-specific double-strand breaks at or near the homing site in intron- or intein-less allele. Subsequently, through nonreciprocal exchange the insertion sequence (intron or intein) is transferred from an intein- or intron-containing allele to an intein- or intron-less allele. The components of host double-strand break repair pathway are thought to finish the "homing" process. Several lines of evidence suggest that homing endonucleases are capable of promoting transposition into ectopic sites within or across genomes for their survival as well as dispersal in natural populations. The occurrence of inteins at high frequencies serves as instructive models for understanding the mechanistic aspects of the process of homing and its evolution. This review focuses on genetic, biochemical, structural, and phylogenetic aspects of homing endonucleases, and their comparison with restriction endonucleases.     

Structural and Functional Characteristics of Homing Endonucleases

Mobile genetic elements constitute a remarkably diverse group of nonessential selfish genes that provide no apparent function to the host. These selfish genes have been implicated in host extinction, speciation and architecture of genetic systems. Homing endonucleases, encoded by the open reading frames embedded in introns or inteins of mobile genetic elements, possess double-stranded DNA-specific endonuclease activity. They inflict sequence-specific double-strand breaks at or near the homing site in intron- or intein-less allele. Subsequently, through nonreciprocal exchange the insertion sequence (intron or intein) is transferred from an intein- or intron-containing allele to an intein- or intron-less allele. The components of host double-strand break repair pathway are thought to finish the “homing” process. Several lines of evidence suggest that homing endonucleases are capable of promoting transposition into ectopic sites within or across genomes for their survival as well as dispersal in natural populations. The occurrence of inteins at high frequencies serves as instructive models for understanding the mechanistic aspects of the process of homing and its evolution. This review focuses on genetic, biochemical, structural, and phylogenetic aspects of homing endonucleases, and their comparison with restriction endonucleases.     

Mitochondrial Intronic Open Reading Frames in Podospora: Mobility and Consecutive Exonic Sequence Variations

The mitochondrial genome of 23 wild-type strains belonging to three different species of the filamentous fungus Podospora was examined. Among the 15 optional sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences. In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group I intronic ORFs are mobile elements and that their transfer, and comcomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes.     

Divalent metal ion differentially regulates the sequential nicking reactions of the GIY-YIG homing endonuclease I-BmoI

Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.     

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.     

The restriction fold turns to the dark side: a bacterial homing endonuclease with a PD-(D/E)-XK motif

The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene-the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease - a fold normally used to protect a bacterial genome from invading DNA through the action of restriction endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH endonucleases - which in turn, are rarely incorporated into restriction/modification systems.     

Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.

A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.     

Catalytic domain structure and hypothesis for function of GIY-YIG intron endonuclease I-TevI

I-TevI, a member of the GIY-YIG family of homing endonucleases, consists of an N-terminal catalytic domain and a C-terminal DNA-binding domain joined by a flexible linker. The GIY-YIG motif is in the N-terminal domain of I-TevI, which corresponds to a phylogenetically widespread catalytic cartridge that is often associated with mobile genetic elements. The crystal structure of the catalytic domain of I-TevI, the first of any GIY-YIG endonuclease, reveals a novel alpha/beta-fold with a central three-stranded antiparallel beta-sheet flanked by three helices. The most conserved and putative catalytic residues are located on a shallow, concave surface and include a metal coordination site. Similarities in the three-dimensional arrangement of the catalytically important residues and the cation-binding site with those of the His-Cys box endonuclease I-PpoI suggest the possibility of mechanistic relationships among these different families of homing endonucleases despite completely different folds.