The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target.

I-TevI, a double-strand DNA endonuclease encoded by the mobile td intron of phage T4, has specificity for the intronless td allele. Genetic and physical studies indicate that the enzyme makes extensive contacts with its DNA substrate over at least three helical turns and around the circumference of the helix. Remarkably, no single nucleotide within a 48 bp region encompassing this interaction domain is essential for cleavage. Although two subdomains (DI and DII) contain preferred sequences, a third domain (DIII), a primary region of contact with the enzyme, displays much lower sequence preference. While DII and DIII suffice for recognition and binding of I-TevI, all three domains are important for formation of a cleavage-competent complex. Mutational, footprinting and interference studies indicate predominant interactions of I-TevI across the minor groove and phosphate backbone of the DNA. Contacts appear not to be at the single nucleotide level; rather, redundant interactions and/or structural recognition are implied. These unusual properties provide a basis for understanding how I-TevI recognizes T-even phage DNA, which is heavily modified in the major groove. These recognition characteristics may increase the range of natural substrates available to the endonuclease, thereby extending the invasive potential of the mobile intron.     

Importance of a single base pair for discrimination between intron-containing and intronless alleles by endonuclease I-BmoI

Homing endonucleases initiate mobility of their host group I introns by binding to and cleaving lengthy recognition sequences that are typically centered on the intron insertion site (IS) of intronless alleles. Because the intron interrupts the endonucleases' recognition sequence, intron-containing alleles are immune to cleavage by their own endonuclease. I-TevI and I-BmoI are related GIY-YIG endonucleases that bind a homologous stretch of thymidylate synthase (TS)-encoding DNA but use different strategies to distinguish intronless from intron-containing substrates. I-TevI discriminates between substrates at the level of DNA binding, as its recognition sequence is centered on the intron IS. I-BmoI, in contrast, possesses a very asymmetric recognition sequence with respect to the intron IS, binds both intron-containing and intronless TS-encoding substrates, but efficiently cleaves only intronless substrate. Here, we show that I-BmoI is extremely tolerant of multiple substitutions around its cleavage sites and has a low specific activity. However, a single G-C base pair, at position -2 of a 39-base pair recognition sequence, is a major determinant for cleavage efficiency and distinguishes intronless from intron-containing alleles. Strikingly, this G-C base pair is universally conserved in phylogenetically diverse TS-coding sequences; this finding suggests that I-BmoI has evolved exquisite cleavage requirements to maximize the potential to spread to variant intronless alleles, while minimizing cleavage at its own intron-containing allele.     

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.     

Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate.

I-TevI is a site-specific, sequence-tolerant intron endonuclease. The crystal structure of the DNA-binding domain of I-TevI complexed with the 20 bp primary binding region of its DNA target reveals an unusually extended structure composed of three subdomains: a Zn finger, an elongated segment containing a minor groove-binding alpha-helix, and a helix-turn-helix. The protein wraps around the DNA, mostly following the minor groove, contacting the phosphate backbone along the full length of the duplex. Surprisingly, while the minor groove-binding helix and the helix-turn- helix subdomain make hydrophobic contacts, the few base-specific hydrogen bonds occur in segments that lack secondary structure and flank the intron insertion site. The multiple base-specific interactions over a long segment of the substrate are consistent with the observed high site specificity in spite of sequence tolerance, while the modular composition of the domain is pertinent to the evolution of homing endonucleases.     

Intron homing with limited exon homology. Illegitimate double-strand-break repair in intron acquisition by phage t4

The td intron of bacteriophage T4 encodes a DNA endonuclease that initiates intron homing to cognate intronless alleles by a double-strand-break (DSB) repair process. A genetic assay was developed to analyze the relationship between exon homology and homing efficiency. Because models predict exonucleolytic processing of the cleaved recipient leading to homologous strand invasion of the donor allele, the assay was performed in wild-type and exonuclease-deficient (rnh or dexA) phage. Efficient homing was supported by exon lengths of 50 bp or greater, whereas more limited exon lengths led to a precipitous decline in homing levels. However, extensive homology in one exon still supported elevated homing levels when the other exon was completely absent. Analysis of these "one-sided" events revealed recombination junctions at ectopic sites of microhomology and implicated nucleolytic degradation in illegitimate DSB repair in T4. Interestingly, homing efficiency with extremely limiting exon homology was greatly elevated in phage deficient in the 3'-5' exonuclease, DexA, suggesting that the length of 3' tails is a major determinant of the efficiency of DSB repair. Together, these results suggest that illegitimate DSB repair may provide a means by which introns can invade ectopic sites.     

SegG endonuclease promotes marker exclusion and mediates co-conversion from a distant cleavage site

Bacteriophages T2 and T4 are closely related T-even phages. However, T4 genetic markers predominate in the progeny of mixed infections, a phenomenon termed marker exclusion. One region previously mapped where the frequency of T2 markers in the progeny is extremely low is located around gene 32. Here, we describe SegG, a GIY-YIG family endonuclease adjacent to gene 32 of phage T4 that is absent from phage T2. In co-infections with T2 and T4, cleavage in T2 gene 32 by T4-encoded SegG initiates a gene conversion event that results in replacement of T2 gene 32 markers with the corresponding T4 sequence. Interestingly, segG inheritance is limited, apparently because of the physical separation of its cleavage and insertion sites, which are 332 base-pairs apart. This contrasts with efficient inheritance of the phage T4 td group I intron and its endonuclease, I-TevI, for which the distance separating the I-TevI cleavage site and td insertion site is 23 base-pairs. Furthermore, we show that co-conversion tracts generated by repair of SegG and I-TevI double-strand breaks contribute to the localized exclusion of T2 markers. Our results demonstrate that the endonuclease activities of SegG and I-TevI promote the spread of these two endonucleases to progeny phage, consistent with their role as selfish genetic elements, and also provide a mechanism by which the genetic contribution of T2 markers to progeny phage is reduced.     

Distribution, sequence homology, and homing of group I introns among T-even-like bacteriophages: evidence for recent transfer of old introns

Self-splicing group I introns are being found in an increasing number of bacteriophages. Most introns contain an open reading frame coding for a homing endo-nuclease that confers mobility to both the intron and the homing endonuclease gene (HEG). The frequent occurrence of intron/HEG has raised questions whether group I introns are spread via horizontal transfer between phage populations. We have determined complete sequences for the known group I introns among T-even-like bacteriophages together with sequences of the intron-containing genes td, nrdB, and nrdD from phages with and without introns. A previously uncharacterized phage isolate, U5, is shown to contain all three introns, the only phage besides T4 found with a "full set" of these introns. Sequence analysis of td and nrdB genes from intron-containing and intronless phages provides evidence that recent horizontal transmission of introns has occurred among the phages. The fact that several of the HEGs have suffered deletions rendering them non-functional implies that the homing endonucleases are of no selective advantage to the phage and are rapidly degenerating and probably dependent upon frequent horizontal transmissions for maintenance within the phage populations. Several of the introns can home to closely related intronless phages during mixed infections. However, the efficiency of homing varies and is dependent on homology in regions flanking the intron insertion site. The occurrence of optional genes flanking the respective intron-containing gene can strongly affect the efficiency of homing. These findings give further insight into the mechanisms of propagation and evolution of group I introns among the T-even-like bacteriophages.     

Intron mobility in the T-even phages: high frequency inheritance of group I introns promoted by intron open reading frames

Intron mobility in the T-even phages has been demonstrated. Efficient nonreciprocal conversion of intron minus (In-) alleles to intron plus (In+) occurred for the td and sunY genes, but not for nrdB. Conversion to In+ was absolutely dependent on expression of the respective intron open reading frame (ORF). Introns were inserted at their cognate sites in an intronless phage genome via an RNA-independent, DNA-based, duplicative recombination event that was stimulated by exon homology. The td intron ORF product directs the endonucleolytic cleavage of DNA, targeting the site of intron integration. A 21 nucleotide deletion of the integration site abolished high frequency intron inheritance. These experiments provide a novel example of gene conversion in prokaryotes, while suggesting a molecular rationale for the inconsistent distribution of introns within highly conserved exon contexts of the T-even phage genomes.     

Strand-specific contacts and divalent metal ion regulate double-strand break formation by the GIY-YIG homing endonuclease I-BmoI

GIY-YIG homing endonucleases are modular enzymes consisting of a well-defined N-terminal catalytic domain connected to a variable C-terminal DNA-binding domain. Previous studies have revealed that the role of the DNA-binding domain is to recognize and bind intronless DNA substrate, positioning the N-terminal catalytic domain such that it is poised to generate a staggered double-strand break by an unknown mechanism. Interactions of the N-terminal catalytic domain with intronless substrate are therefore a critical step in the reaction pathway but have been difficult to define. Here, we have taken advantage of the reduced activity of I-BmoI, an isoschizomer of the well-studied bacteriophage T4 homing endonuclease I-TevI, to examine double-strand break formation by I-BmoI. We present evidence demonstrating that I-BmoI generates a double-strand break by two sequential but chemically independent nicking reactions where divalent metal ion is a limiting factor in top-strand nicking. We also show by in-gel footprinting that contacts by the I-BmoI catalytic domain induce significant minor groove DNA distortions that occur independently of bottom-strand nicking. Bottom-strand contacts are critical for accurate top-strand nicking, whereas top-strand contacts have little influence on the accuracy of bottom-strand nicking. We discuss our results in the context of current models of GIY-YIG endonuclease function, with emphasis on the role of divalent metal ion and strand-specific contacts in regulating the activity of a single active site to generate a staggered double-strand break.     

Perpetuating the homing endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference

Homing endonucleases are sequence-tolerant DNA endonucleases that act as mobile genetic elements. The ability of homing endonucleases to cleave substrates with multiple nucleotide substitutions suggests a high degree of adaptability in that changing or modulating cleavage preference would require relatively few amino acid substitutions. Here, using directed evolution experiments with the GIY-YIG homing endonuclease I-TevI that targets the thymidylate synthase gene of phage T4, we readily isolated variants that dramatically broadened I-TevI cleavage preference, as well as variants that fine-tuned cleavage preference. By combining substitutions, we observed an ∼10 000-fold improvement in cleavage on some substrates not cleaved by the wild-type enzyme, correlating with a decrease in readout of information content at the cleavage site. Strikingly, we were able to change the cleavage preference of I-TevI to that of the isoschizomer I-BmoI which targets a different cleavage site in the thymidylate synthase gene, recapitulating the evolution of cleavage preference in this family of homing endonucleases. Our results define a strategy to isolate GIY-YIG nuclease domains with distinct cleavage preferences, and provide insight into how homing endonucleases may escape a dead-end life cycle in a population of saturated target sites by promoting transposition to different target sites.     

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.     

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.     

Purification and characterization of the SegA protein of bacteriophage T4, an endonuclease related to proteins encoded by group I introns.

Although not encoded by an intron, the bacteriophage T4 SegA protein shares common amino acid motifs with a family of proteins found within mobile group I introns present in fungi and phage. Each of these intron-encoded proteins is thought to initiate the homing of its own intron by cleaving the intronless DNA at or near the site of insertion. Previously, we have found that SegA also cleaves DNA. In this report, we have purified the SegA protein and characterized this endonuclease activity extensively. SegA protein cleaved circular and linear plasmids, DNA containing unmodified cytosines, and wild-type T4 DNA containing hydroxymethylated, glucosylated cytosines. In all cases, certain sites on the DNA were highly preferred for cleavage, but with increasing protein concentration or time of incubation, cleavage occurred at many sites. SegA cleaving activity was stimulated by the presence of ATP or ATP gamma S. Sequence analysis of three highly preferred cleavage sites did not reveal a simple consensus sequence, suggesting that even among highly preferred sites, SegA tolerates many different sequences. A T4 segA amber mutant that we constructed had no phenotype, and PCR analyses indicated that several T-even-related phages lack the segA gene. Taken together, our results show that SegA is an endonuclease with a hierarchy of site specificity, and these results are consistent with the insertion of segA DNA into the T4 genome some time after the divergence of the closely consistent with the insertion of segA DNA into the T4 genome some time after the divergence of the closely related T-even phages.     

Conserved target for group II intron insertion in relaxase genes of conjugative elements of gram-positive bacteria

The lactococcal group II intron Ll.ltrB interrupts the ltrB relaxase gene within a region that encodes a conserved functional domain. Nucleotides essential for the homing of Ll.ltrB into an intronless version of ltrB are found exclusively at positions required to encode amino acids broadly conserved in a family of relaxase proteins of gram-positive bacteria. Two of these relaxase genes, pcfG from the enterococcal plasmid pCF10 and the ORF4 gene in the streptococcal conjugative transposon Tn5252, were shown to support Ll.ltrB insertion into the conserved motif at precisely the site predicted by sequence homology with ltrB. Insertion occurred through a mechanism indistinguishable from retrohoming. Splicing and retention of conjugative function was demonstrated for pCF10 derivatives containing intron insertions. Ll.ltrB targeting of a conserved motif of a conjugative element suggests a mechanism for group II intron dispersal among bacteria. Additional support for this mechanism comes from sequence analysis of the insertion sites of the E.c.I4 family of bacterial group II introns.     

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.     

Characterization of homing endonucleases

Homing endonucleases are a class of site-specific DNA endonucleases encoded by open reading frames within introns and inteins. They initiate the mobility of their host element by recognizing intronless or inteinless alleles of their host gene and making a double-strand break. The homing endonucleases are notable for their long target sites and a tolerance for sequence polymorphisms in their substrates. The methods used to study homing endonucleases are similar to those used to study protein-DNA interactions in general. However, some variations and specialized techniques are useful in characterizing homing endonucleases and these methods are discussed.     

Structure of Bacteriophage T4 Endonuclease II Mutant E118A, a Tetrameric GIY-YIG Enzyme

Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16 kDa, 136 aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P2₁ with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context.