Intron-encoded Endonuclease Research Articles

Physical and gene maps of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome

A physical map of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome has been constructed with restriction endonucleases PmeI, SwaI, and an intron-encoded endonuclease I-CeuI. The estimated size of the genome is 2.7 Mb. On the genome 49 genes or operons have been mapped. Two rRNA operons are separated by 600 kb and transcribed oppositely.

Selfish DNA: The best defense is a good offense

The recent discovery of novel biochemical activities of intron-encoded endonucleases emphasizes the selfish nature of mobile genetic elements.

Substrate binding and turnover by the highly specific I-PpoI endonuclease.

Intron-encoded endonucleases are distinguished by their ability to catalyze the cleavage of double-stranded DNA with high specificity. I-PpoI endonuclease, an intron-encoded endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) that catalyzes the cleavage of a large asymmetric DNA sequence (15 base pairs). Here, the interactions of I-PpoI with its substrate were examined during both binding (in the absence of Mg2+) and catalysis (in the presence of Mg2+). Using circular permutation assays, I-PpoI was shown to bend its substrate by 38 +/- 4 degrees upon binding. Two independent methods, gel mobility shift assays and fluorescence polarization assays, revealed that I-PpoI binds tightly to its substrate. Values of Kd range from 3.3 to 112 nM, increasing with increasing NaCl concentration. Similar salt effects on the values of Km were observed during steady-state catalysis. At low salt concentrations, the value of kcat/Km for the cleavage of an oligonucleotide duplex approaches 10(8) M-1 s-1. Although other divalent cations can replace Mg2+, catalysis by I-PpoI is most efficient in the presence of an oxophilic metal ion that prefers an octahedral geometry: Mg2+ > Mn2+ > Ca2+ = Co2+ > Ni2+ > Zn2+. Together, these results provide the first chemical insight into substrate binding and turnover by an intron-encoded endonuclease.

Intron-encoded Endonuclease I- TevII Binds Across the Minor Groove and Induces Two Distinct Conformational Changes in its DNA Substrate

I- TevII is the homing endonuclease encoded by the sunY intron of bacteriophage T4. The enzyme cleaves an intronless sunY gene near the exon I-exon II junction, thereby initiating intron homing into its cognate intronless allele. Specifically, I- TevII cleaves its DNA target 13 to 15 nucleotides (nt) downstream of the sunY intron insertion site, generating 2-nt 3′-OH extensions. Here, we present evidence that I- TevII makes predominantly minor groove contacts in two regions of its recognition sequence, as does I- TevI, the other homing endonuclease encoded by phage T4. Following cleavage, I- TevII was shown to remain bound to one of its DNA products, suggesting possible additional roles for the endonuclease in the mobility process. Interestingly, two distinct conformational changes were detected by gel analysis in the DNA substrate following binding by I- TevII, one occurring in the absence of Mg 2+, the second being dependent on the presence of Mg 2+. The Mg 2+-induced distortion accompanies a nick in one strand, and may serve to bring the cleavage site on the other strand into proximity with the catalytic domain of the protein.

Rapid Restriction Mapping of Cosmids by Sequence-Specific Triple-Helix-Mediated Affinity Capture

A simple and rapid strategy for restriction mapping based on sequence-specific triple-helix affinity capture (TAC) was developed. The strategy was applied to the analysis of cosmid clones by the construction of a new cosmid vector, ScosTriplex-II, containing two different triple-helix-forming sequences flanking the cloning site of the original SuperCos-1 cosmid vector. For restriction mapping, the recombinant cosmid DNA is digested withNotI restriction enzyme or with one of four intron-encoded endonucleases for excision of intact inserts followed by controlled partial digestion with a mapping enzyme used in conjunction with the corresponding methyltransferase. The partial digestion products are combined with biotinylated triple-helix-forming oligonucleotides to form a triple-helical complex. The triple-helix complexes are immobilized on streptavidin-coated magnetic beads, washed, and eluted with pH 9 buffer solution. The fragments are separated and directly sized by agarose gel electrophoresis. Bidirectional maps are obtained simultaneously by binding to the two different triple-helix-forming oligonucleotides. No probe labeling, gel drying, blotting to membranes, hybridization, or autoradiography is necessary. Also, TAC conditions that permit gel-free isolation of the terminal restriction fragments from cosmid inserts were found. These advantages afforded by ScosTriplex-II should facilitate the automation of cosmid restriction site fingerprinting needed for large-scale mapping and sequencing projects.

Mechanisms of Intron Mobility

Mechanisms of Intron Mobility

Intron-encoded endonuclease I-TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site.

I-TevI, the intron-encoded endonuclease from the thymidylate synthase (td) gene of bacteriophage T4, binds its DNA substrate across the minor groove in a sequence-tolerant fashion. We demonstrate here that the 28 kDa I-TevI binds the extensive 37 bp td homing site as a monomer and significantly distorts its substrate. In situ cleavage assays and phasing analyses indicate that upon nicking the bottom strand of the td homing site, I-TevI induces a directed bend of 38 degrees towards the major groove near the cleavage site. Formation of the bent I-TevI-DNA complex is proposed to promote top-strand cleavage of the homing site. Furthermore, reductions in the degree of distortion and in the efficiency of binding base-substitution variants of the td homing site indicate that sequences flanking the cleavage site contribute to the I-TevI-induced conformational change. These results, combined with genetic, physical and computer-modeling studies, form the basis of a model, wherein I-TevI acts as a hinged monomer to induce a distortion that widens the minor groove, facilitating access to the top-strand cleavage site. The model is compatible with both unmodified DNA and glucosylated hydroxymethylcytosine-containing DNA, as exists in the T-even phages.

Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae.

The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.

Rearrangements in the genome of the bacterium Salmonella typhi.

We have determined the genomic map of the bacterium Salmonella typhi Ty2, the causal organism of typhoid fever, by using pulsed-field gel electrophoresis. Digestion of the Ty2 genome with endonucleases Xba I, Bln I, and Ceu I yielded 33, 26, and 7 fragments, respectively, that were placed in order on a circular chromosome of 4780 kb. Transposon Tn10 was inserted in specific genes of Salmonella typhimurium and transduced into S. typhi, and thus, the positions of 37 S. typhi genes were located through the Xba I and Bln I sites of the Tn10. Gene order on chromosomes of Escherichia coli K-12 and S. typhimurium LT2 is remarkably conserved; however, the gene order in S. typhi Ty2 is different, suggesting it has undergone major genomic rearrangements during its evolution. These rearrangements include inversions and transpositions in the 7 DNA fragments between the seven rrn operons for rRNA (postulated to be due to homologous recombination in these rrn genes), another inversion that covers the replication terminus region (resembling inversions found in other enteric bacteria), and at least three insertions, one as large as 118 kb. Partial digestion of genomic DNA with the intron-encoded endonuclease I-Ceu I, which cuts only in rrn genes, shows chromosomal rearrangements, apparently due to homologous recombination in the rrn genes, that were detected in all wild-type strains of S. typhi tested. These rearrangements may have been selected to compensate for the insertions that otherwise would have altered the locations of genes with respect to the origin and terminus of replication. These observations are relevant to our view of the evolution of the bacterial genome and may be significant in the virulence of S. typhi.

Homing sweet homing: mobile introns in bacterial viruses

Several bacteriophage group I introns are self-splicing at the RNA level, while they behave as mobile genetic elements at the DNA level. Contained within the catalytic RNAs are open reading frames which encode endonucleases that initiate the intron mobility reaction. This process, called homing, is a gene conversion event whereby an intron transfers from an intron-plus allele to the cognate intron-minus allele. Such mobile introns are phylogenetically diverse, being found in both prokaryotic and eukaryotic genomes. In this context, the accessibility of the phage systems and the unusual nature of the phage endonuclease:target site interactions make them a rewarding focus of study. We will discuss the mobility process and the hierarchy of relationships between the bacteriophage, its group I introns and the intron-encoded endonucleases.

Evolution of mobile group I introns: recognition of intron sequences by an intron-encoded endonuclease.

Mobile group I introns are hypothesized to have arisen after invasion by endonuclease-encoding open reading frames (ORFs), which mediate their mobility. Consistent with an endonuclease-ORF invasion event, we report similarity between exon junction sequences (the recognition site for the mobility endonuclease) and intron sequences flanking the endonuclease ORF in the sunY gene of phage T4. Furthermore, we have demonstrated the ability of the intron-encoded endonuclease to recognize and cleave these intron sequences when present in fused form in synthetic constructs. These observations and accompanying splicing data are consistent with models in which the invading endonuclease ORF is provided safe haven within a splicing element. In turn the intron is afforded immunity to the endonuclease product, which imparts mobility to the intron.

Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.

Double-strand breaks introduced into DNA in vivo have been shown to enhance homologous recombination in a variety of chromosomal and extrachromosomal loci in Saccharomyces cerevisiae. To introduce double-strand breaks in DNA at defined locations in mammalian cells, we have constructed a mammalian expression vector for a modified form of I-Sce I, a yeast mitochondrial intron-encoded endonuclease with an 18-bp recognition sequence. Expression of the modified I-Sce I endonuclease in COS1 cells results in cleavage of model recombination substrates and enhanced extrachromosomal recombination, as assayed by chloramphenicol acetyltransferase activity and Southern blot analysis. Constitutive expression of the endonuclease in mouse 3T3 cells is not lethal, possibly due to either the lack of I-Sce I sites in the genome or sufficient repair of them. Expression of an endonuclease with such a long recognition sequence will provide a powerful approach to studying a number of molecular processes in mammalian cells, including homologous recombination.

Interaction of the intron-encoded mobility endonuclease I-PpoI with its target site.

Endonucleases encoded by mobile group I introns are highly specific DNases that induce a double-strand break near the site to which the intron moves. I-PpoI from the acellular slime mold Physarum polycephalum mediates the mobility of intron 3 (Pp LSU 3) in the extrachromosomal nuclear ribosomal DNA of this organism. We showed previously that cleavage by I-PpoI creates a four-base staggered cut near the point of intron insertion. We have now characterized several further properties of the endonuclease. As determined by deletion analysis, the minimal target site recognized by I-PopI was a sequence of 13 to 15 bp spanning the cleavage site. The purified protein behaved as a globular dimer in sedimentation and gel filtration. In gel mobility shift assays in the presence of EDTA, I-PpoI formed a stable and specific complex with DNA, dissociating with a half-life of 45 min. By footprinting and interference assays with methidiumpropyl-EDTA-iron(II), I-PpoI contacted a 22- to 24-bp stretch of DNA. The endonuclease protected most of the purines found in both the major and minor grooves of the DNA helix from modification by dimethyl sulfate (DMS). However, the reactivity to DMS was enhanced at some purines, suggesting that binding leads to a conformational change in the DNA. The pattern of DMS protection differed fundamentally in the two partially symmetrical halves of the recognition sequence.

Interaction of the Intron-Encoded Mobility Endonuclease I-PpoI with its Target Site

Endonucleases encoded by mobile group I introns are highly specific DNases that induce a double-strand break near the site to which the intron moves. I-PpoI from the acellular slime mold Physarum polycephalum mediates the mobility of intron 3 (Pp LSU 3) in the extrachromosomal nuclear ribosomal DNA of this organism. We showed previously that cleavage by I-PpoI creates a four-base staggered cut near the point of intron insertion. We have now characterized several further properties of the endonuclease. As determined by deletion analysis, the minimal target site recognized by I-PopI was a sequence of 13 to 15 bp spanning the cleavage site. The purified protein behaved as a globular dimer in sedimentation and gel filtration. In gel mobility shift assays in the presence of EDTA, I-PpoI formed a stable and specific complex with DNA, dissociating with a half-life of 45 min. By footprinting and interference assays with methidiumpropyl-EDTA-iron(II), I-PpoI contacted a 22- to 24-bp stretch of DNA. The endonuclease protected most of the purines found in both the major and minor grooves of the DNA helix from modification by dimethyl sulfate (DMS). However, the reactivity to DMS was enhanced at some purines, suggesting that binding leads to a conformational change in the DNA. The pattern of DMS protection differed fundamentally in the two partially symmetrical halves of the recognition sequence.

Asymmetrical recognition and activity of the I-SceI endonuclease on its site and on intron-exon junctions.

Group I intron-encoded endonucleases represent a new class of double strand cutting endonucleases whose function is to initiate the homing of introns by generating double strand breaks in site-specific sequences. We have studied the mechanism of interaction of the I-SceI endonuclease with different DNA substrates derived from its natural site in the intron-less gene or from intron-exon junctions in the gene with an intron. We show that the enzyme recognizes its asymmetrical site with high affinity binding to the sequence corresponding to the downstream exon followed by binding to the upstream exon and catalysis of phosphodiester bond hydrolysis. Asymmetrical nicking activity is observed as an intermediate of the cleavage reaction. In the intron-containing gene, the enzyme recognizes the downstream intron-exon junction without any cleavage activity. This binding raises the possibility of a specific function of homing endonucleases in either gene expression or intron homing steps subsequent to DNA cleavage.

Comprehensive restriction enzyme lists to update any DNA sequence computer program

Restriction enzyme lists are presented for the practical working geneticist to update any DNA computer program. These lists combine formerly scattered information and contain all presently known restriction enzymes with a unique recognition sequence, a cut site, or methylation (in)sensitivity. The lists are in the shortest possible form to also be functional with small DNA computer programs, and will produce clear restriction maps without any redundancy or loss of information. The lists discern between commercial and noncommercial enzymes, and prototype enzymes and different isoschizomers are cross-referenced. Differences in general methylation sensitivities and (in)sensitivities against Dam and Dcm methylases of Escherichia coli are indicated. Commercial methylases and intron-encoded endonucleases are included. An address list is presented to contact commercial suppliers. The lists are constantly updated and available in electronic form as pure US ASCII files, and in formats for the DNA computer programs DNA-Strider for Apple Macintosh, and DNAsis for IBM personal computers or compatibles via e-mail from the internet address: netservembl-heidelberg.de by sending only the message help relibrary.

I-Sce III an intron-encoded DNA endonuclease from yeast mitochondria. Asymmetrical DNA binding properties and cleavage reaction.

We have previously discovered the new intron-encoded endonuclease I-Sce III by expressing, in E. coli, the ORF contained in the third intron of the yeast mitochondrial COX I gene. In this work, we analyzed the in vitro properties of partially purified I-Sce III and found that it is a very specific DNA endonuclease, tolerating relatively few base changes in its 20 base pair long target site. I-Sce III should be a useful molecular tool to analyze the structure of large genomes. Interestingly, I-Sce III is the first P1-P2 DNA endonuclease for which DNA binding properties could be analyzed by band-shift experiments. Clearly, the cleavage products corresponding to the upstream A3 exon and to the downstream A4 exon could compete with the substrate A3-A4 in forming a DNA-protein complex. However, the A3 exon competes more efficiently than the downstream A4 product. The cleavage of the two DNA strands is also asymmetric the top strand (non-transcribed strand) is cleaved faster than the bottom strand, a property found under various experimental conditions. These findings suggest that this intron-encoded DNA endonuclease may have role in the RNA splicing process of the intron.

I-Sce III: a novel group I intron-encoded endonuclease from the yeast mitochondria.

I-Sce III: a novel group I intron-encoded endonuclease from the yeast mitochondria.

A new method for specific cleavage of megabase-size chromosomal DNA by lambda-terminase.

The development of methods for cleavage of DNA at specific site(s) that are widely spaced would facilitate physical mapping of large genomes. Several methods for rare and specific cleavage of chromosomal DNAs require a nearly complete methylation of a given type of restriction site except the one that is specifically protected. It is expected that as the target DNA increases in length, it will become less likely to achieve nearly complete methylation. The intron-encoded endonucleases may also provide a capability to cleave megabase-sized DNA segments due to their very large recognition sequences. However, there are endogenous cleavage sites in the chromosomes of most organisms. We present here a new method to specifically cleave intact chromosomal DNA using lambda-terminase. A plasmid containing two specific cleavage sites (cohesive-end sites) for lambda-terminase was specifically introduced into the E.coli genome and into chromosome V of S.cerevisiae. Chromosomal DNA was prepared from the resulting strains, and then cleaved with lambda-terminase. The results showed that the 4.7-megabase pair (Mb) circular E.coli chromosome and the 0.58-Mb linear yeast chromosome V were specifically cleaved at the desired sites with very high efficiencies. The approach of using the lambda-terminase cleavage reaction is a simple one-step procedure with a high specificity which is particularly suitable for mapping very large genomes of eucaryotes.

Trans and cis requirements for intron mobility in a prokaryotic system.

Intron mobility requires cleavage of an intronless allele by an intron-encoded endonuclease, followed by transfer of the intron into the cleaved recipient. The mobile phage introns provide an opportunity to identify accessory functions involved in the intron inheritance process. To test for trans and cis requirements of mobility in Escherichia coli, we have exploited the td intron of phage T4 in both phage T4 and lambda genetic backgrounds. Mobility depends on host or phage recombinase functions, RecA or UvsX, respectively. The process also requires a phage-encoded 5'----3' exonuclease activity and associated annealing function that can be provided by phage lambda. Finally, host-encoded 3'----5' exonuclease activities are also implicated in intron inheritance. We demonstrated further that restriction enzymes could substitute for the intron-encoded endonuclease, indicating that the endonuclease does not have an essential role in recombination. Neither the precise position nor the nature of the double-strand break was critical to intron transfer. These features provide insight into the recombination pathway and are factors impacting on the spread of introns throughout natural populations.