The group I intron of apocytochrome b gene from Chlamydomonas smithii encodes a site-specific endonuclease

The mitochondrial DNA molecules of two interfertile algal species, Chlamydomonas smithii and C. reinhardtii, are co-linear except for a 1075 bp intron (the -insert) that is present in the cob gene of C. smithii. The -insert, a group I intron (Cs cob·1) containing an open reading frame (ORF) which encodes a basic, hydrophilic protein of 237 amino acids, is unidirectionally transmitted to all diploid progeny during interspecific crosses. In this report, we show that the Cs cob·1-encoded protein is a site-specific endonuclease (I-Csm I) which could mediate the intron transfer via the gene conversion mechanism. The Cs cob·1 ORF was cloned into the vector pMALcr1 and over-expressed as a hybrid protein fused to maltose-binding protein (MBP). This fusion protein exhibited an in vivo endonuclease activity which specifically cleaved the intron homing site within the intronless cob gene.     

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.     

Crystallization and preliminary x-ray studies of I-CreI: a group I intron-encoded endonuclease from C. reinhardtii

Group I intron endonuclease I-CreI is encoded by an open reading frame contained within a self-splicing intron in the Chlamydomonas reinhardtii chloroplast 23S rRNA gene. I-CreI initiates the lateral transfer or homing of this intron by specifically recognizing and cleaving a pseudopalindromic 19–24 bp homing site in chloroplast 23S rRNA genes that lack the intron. The gene encoding this enzyme has been subcloned, and the protein product has been purified and crystallized. The crystals belong to space group P321, with unit cell dimensions a = b = 78.2 Å, c = 67.4 Å. The crystal unit cell is consistent with an asymmetric unit consisting of the enzyme monomer. The specific volume of this unit cell is 3.3 ų/Da. The crystals diffract to at least 3.0 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. © 1997 Wiley-Liss Inc.     

Mutations altering the cleavage specificity of a homing endonuclease

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein–DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences.     

A maturase that specifically stabilizes and activates its cognate group I intron at high temperatures

Folding of large structured RNAs into their functional tertiary structures at high temperatures is challenging. Here we show that I-TnaI protein, a small LAGLIDADG homing endonuclease encoded by a group I intron from a hyperthermophilic bacterium, acts as a maturase that is essential for the catalytic activity of this intron at high temperatures and physiological cationic conditions. I-TnaI specifically binds to and induces tertiary packing of the P4-P6 domain of the intron; this RNA-protein complex might serve as a thermostable platform for active folding of the entire intron. Interestingly, the binding affinity of I-TnaI to its cognate intron RNA largely increases with temperature; over 30-fold stronger binding at higher temperatures relative to 37 °C correlates with a switch from an entropy-driven (37 °C) to an enthalpy-driven (55-60 °C) interaction mode. This binding mode may represent a novel strategy how an RNA binding protein can promote the function of its target RNA specifically at high temperatures.     

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

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

Purification and characterization of the DNA cleavage and recognition site of I-ScaI mitochondrial group I intron encoded endonuclease produced in Escherichia coli

The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs. Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain. It acts as a specific homing endonuclease I-ScaI promoting intron mobility and as a maturase promoting intron splicing. Here we describe the synthesis of a universal code equivalent to the mitochondrial sequence coding for this protein and the in vitro characterization of I-ScaI endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli. We have also determined the cleavage pattern as well as the recognition site of p28bi2. It was found that p28bi2 generates a double-strand cleavage downstream from the intron insertion site with 4 nt long 3′-overhangs. Mutational analysis of the DNA target site shows that p28bi2 recognizes a 16–19 bp sequence from positions –11 to +8 with respect to the intron insertion site.     

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.     

I-BasI and I-HmuI: two phage intron-encoded endonucleases with homologous DNA recognition sequences but distinct DNA specificities

I-HmuI and I-BasI are two highly similar nicking DNA endonucleases, which are each encoded by a group I intron inserted into homologous sites within the DNA polymerase genes of Bacillus phages SPO1 and Bastille, respectively. Here, we present a comparison of the DNA specificities and cleavage activities of these enconucleases with homologous target sites. I-BasI has properties that are typical of homing endonucleases, nicking the intron-minus polymerase genes in either host genome, three nucleotides downstream of the intron insertion site. In contrast, I-HmuI nicks both the intron-plus and intron-minus site in its own host genome, but does not act on the target from Bastille phage. Although the enzymes have distinct DNA substrate specificities, both bind to an identical 25bp region of their respective intron-minus DNA polymerase genes surrounding the intron insertion site. The endonucleases appear to interact with the DNA substrates in the downstream exon 2 in a similar manner. However, whereas I-HmuI is known to make its only base-specific contacts within this exon region, structural modeling analyses predict that I-BasI might make specific base contacts both upstream and downstream of the site of intron insertion. The predicted requirement for base-specific contacts in exon 1 for cleavage by I-BasI was confirmed experimentally. This explains the difference in substrate specificities between the two enzymes, including the observation that the former enzyme is relatively insensitive to the presence of an intron upstream of exon 2. These differences are likely a consequence of divergent evolutionary constraints.     

The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility

Background

The a2 mating type locus gene lga2 is critical for uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. Specifically, the absence of lga2 results in biparental inheritance, along with efficient transfer of intronic regions in the large subunit rRNA gene between parental molecules. However, the underlying role of the predicted LAGLIDADG homing endonuclease gene I-UmaI located within the group II intron LRII1 has remained unresolved.

Methodology/Principal Findings

We have investigated the enzymatic activity of I-UmaI in vitro based on expression of a tagged full-length and a naturally occurring mutant derivative, which harbors only the N-terminal LAGLIDADG domain. This confirmed Mg²⁺-dependent endonuclease activity and cleavage at the LRII1 insertion site to generate four base pair extensions with 3′ overhangs. Specifically, I-UmaI recognizes an asymmetric DNA sequence with a minimum length of 14 base pairs (5′-GACGGGAAGACCCT-3′) and tolerates subtle base pair substitutions within the homing site. Enzymatic analysis of the mutant variant indicated a correlation between the activity in vitro and intron homing. Bioinformatic analyses revealed that putatively functional or former functional I-UmaI homologs are confined to a few members within the Ustilaginales and Agaricales, including the phylogenetically distant species Lentinula edodes, and are linked to group II introns inserted into homologous positions in the LSU rDNA.

Conclusions/Significance

The present data provide strong evidence that intron homing efficiently operates under conditions of biparental inheritance in U. maydis. Conversely, uniparental inheritance may be critical to restrict the transmission of mobile introns. Bioinformatic analyses suggest that I-UmaI-associated introns have been acquired independently in distant taxa and are more widespread than anticipated from available genomic data.     

Coincidence of cleavage sites of intron endonuclease I-TevI and critical sequences of the host thymidylate synthase gene

To maximize spread of their host intron or intein, many homing endonucleases recognize nucleotides that code for important and conserved amino acid residues of the target gene. Here, we examine the cleavage requirements for I-TevI, which binds a stretch of thymidylate synthase (TS) DNA that codes for functionally critical residues in the TS active site. Using an in vitro selection scheme, we identified two base-pairs in the I-TevI cleavage site region as important for cleavage efficiency. These were confirmed by comparison of I-TevI cleavage efficiencies on mutant and on wild-type substrates. We also showed that nicking of the bottom strand by I-TevI is not affected by mutation of residues surrounding the bottom-strand cleavage site, unlike other homing endonucleases. One of these two base-pairs is universally conserved in all TS sequences, and is identical with a previously identified cleavage determinant of I-BmoI, a related GIY-YIG endonuclease that binds a homologous stretch of TS-encoding DNA. The other base-pair is conserved only in a subset of TS genes that includes the I-TevI, but not the I-BmoI, target sequence. Both the I-TevI and I-BmoI cleavage site requirements correspond to functionally critical residues involved in an extensive hydrogen bond network within the TS active site. Remarkably, these cleavage requirements correlate with TS phylogeny in bacteria, suggesting that each endonuclease has individually adapted to efficiently cleave distinct TS substrates.     

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

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

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

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

Crystal Structure of the Homing Endonuclease I-CvuI Provides a New Template for Genome Modification

Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage even in complete genomes. Although a large number of homing endonucleases have been identified, the landscape of possible target sequences is still very limited to cover the complexity of the whole eukaryotic genome. Therefore, the finding and molecular analysis of homing endonucleases identified but not yet characterized may widen the landscape of possible target sequences. The previous characterization of protein-DNA interaction before the engineering of new homing endonucleases is essential for further enzyme modification. Here we report the crystal structure of I-CvuI in complex with its target DNA and with the target DNA of I-CreI, a homologue enzyme widely used in genome engineering. To characterize the enzyme cleavage mechanism, we have solved the I-CvuI DNA structures in the presence of non-catalytic (Ca²⁺) and catalytic ions (Mg²⁺). We have also analyzed the metal dependence of DNA cleavage using Mg²⁺ ions at different concentrations ranging from non-cleavable to cleavable concentrations obtained from in vitro cleavage experiments. The structure of I-CvuI homing endonuclease expands the current repertoire for engineering custom specificities, both by itself as a new scaffold alone and in hybrid constructs with other related homing endonucleases or other DNA-binding protein templates.     

Biochemical characterization of I-CmoeI reveals that this H-N-H homing endonuclease shares functional similarities with H-N-H colicins

Endonuclease assays of the H-N-H proteins encoded by two group I introns in the Chlamydomonas moewusii chloroplast psbA gene revealed that the CmpsbA·1 intron specifies a site-specific DNA endonuclease, designated I-CmoeI. Like most previously reported intron-encoded endonucleases, I-CmoeI generates a double-strand break near the insertion site of its encoding intron, leaving 3′ extensions of 4 nt. This enzyme was purified from Escherichia coli as a fusion protein with a His tag at its N-terminus. The recombinant protein (rI-CmoeI) requires a divalent alkaline earth cation for DNA cleavage (Mg²⁺ > Ca²⁺ > Sr²⁺ > Ba²⁺). It also requires a metal cofactor for DNA binding, a property shared with H-N-H colicins but not with the homing endonucleases characterized to date. rI-CmoeI binds its recognition sequence as a monomer, as revealed by gel retardation assays. K_m and k_cat values of 100 ± 40 pM and 0.26 ± 0.04 min^–1, respectively, were determined. Replacement of the first histidine of the H-N-H motif by an alanine residue abolishes both rI-CmoeI activity and binding to its substrate. We propose that this conserved histidine residue plays a role in binding the metal cofactor and that such binding induces a structural modification of the enzyme which is required for DNA recognition.     

Homing endonuclease I-TevIII: dimerization as a means to a double-strand break

Homing endonucleases are unusual enzymes, capable of recognizing lengthy DNA sequences and cleaving site-specifically within genomes. Many homing endonucleases are encoded within group I introns, and such enzymes promote the mobility reactions of these introns. Phage T4 has three group I introns, within the td, nrdB and nrdD genes. The td and nrdD introns are mobile, whereas the nrdB intron is not. Phage RB3 is a close relative of T4 and has a lengthier nrdB intron. Here, we describe I-TevIII, the H–N–H endonuclease encoded by the RB3 nrdB intron. In contrast to previous reports, we demonstrate that this intron is mobile, and that this mobility is dependent on I-TevIII, which generates 2-nt 3′ extensions. The enzyme has a distinct catalytic domain, which contains the H–N–H motif, and DNA-binding domain, which contains two zinc fingers required for interaction with the DNA substrate. Most importantly, I-TevIII, unlike the H–N–H endonucleases described so far, makes a double-strand break on the DNA homing site by acting as a dimer. Through deletion analysis, the dimerization interface was mapped to the DNA-binding domain. The unusual propensity of I-TevIII to dimerize to achieve cleavage of both DNA strands underscores the versatility of the H–N–H enzyme family.     

Coevolution of a homing endonuclease and its host target sequence

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