I-PfoP3I: a novel nicking HNH homing endonuclease encoded in the group I intron of the DNA polymerase gene in Phormidium foveolarum phage Pf-WMP3.
ABSTRACT: Homing endonucleases encoded in a group I self-splicing intron in a protein-coding gene in cyanophage genomes have not been reported, apart from some free-standing homing edonucleases. In this study, a nicking DNA endonuclease, I-PfoP3I, encoded in a group IA2 intron in the DNA polymerase gene of a T7-like cyanophage Pf-WMP3, which infects the freshwater cyanobacterium Phormidium foveolarum is described. The Pf-WMP3 intron splices efficiently in vivo and self-splices in vitro simultaneously during transcription. I-PfoP3I belongs to the HNH family with an unconventional C-terminal HNH motif. I-PfoP3I nicks the intron-minus Pf-WMP3 DNA polymerase gene more efficiently than the Pf-WMP4 DNA polymerase gene that lacks any intervening sequence in vitro, indicating the variable capacity of I-PfoP3I. I-PfoP3I cleaves 4 nt upstream of the intron insertion site on the coding strand of EXON 1 on both intron-minus Pf-WMP3 and Pf-WMP4 DNA polymerase genes. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a 14 bp region downstream of the cut site. I-PfoP3I requires Mg(2+), Ca(2+) or Mn(2+) for nicking activity. Phylogenetic analysis suggests that the intron and homing endonuclease gene elements might be inserted in Pf-WMP3 genome individually after differentiation from Pf-WMP4. To our knowledge, this is the first report of the presence of a group I self-splicing intron encoding a functional homing endonuclease in a protein-coding gene in a cyanophage genome.
Project description:Homing endonucleases are site-specific DNA endonucleases that typically function as mobile genetic elements by introducing a double-strand break (DSB) in genomes that lack the endonuclease, resulting in a unidirectional gene conversion event that mobilizes the homing endonuclease gene and flanking DNA. Here, we characterize phage T4-encoded mobE, a predicted free-standing HNH family homing endonuclease. We show that mobE is promoterless and dependent on upstream transcription for expression, and that an internal intrinsic terminator regulates mobE transcript levels. Crucially, in vivo mapping experiments revealed a MobE-dependent, strand-specific nick in the non-coding strand of the nrdB gene of phage T2. An internal deletion of the predicted HNH catalytic motif of MobE abolishes nicking, and reduces high-frequency inheritance of mobE. Sequence polymorphisms of progeny phage that inherit mobE are consistent with DSB repair pathways. Significantly, we found that mobility of the neighboring I-TevIII, a defunct homing endonuclease encoded within a group I intron interrupting the nrdB gene of phage T4, was dependent on an intact mobE gene. Thus, our data indicate that the stagnant nrdB intron and I-TevIII are mobilized in trans as a consequence of a MobE-dependent gene conversion event, facilitating persistence of genetic elements that have no inherent means of promoting their own mobility.
Project description:Mobile group I introns are RNA splicing elements that have been invaded by endonuclease genes. These endonucleases facilitate intron mobility by a unidirectional, duplicative gene-conversion process known as homing . Survival of the invading endonuclease depends upon its ability to promote intron mobility. Therefore, the endonuclease must either quickly change its cleavage specificity to match the site of intron insertion, or it must already be preadapted to cleave this sequence. Here we show that the group I intron in the DNA polymerase gene of T7-like bacteriophage PhiI is mobile, dependent upon its intronic HNH homing endonuclease gene, I-TslI. We also show that gene 5.3 of phage T3, located adjacent to its intronless DNA polymerase gene, is a homologous homing endonuclease gene whose protein product initiates efficient spread of gene 5.3 into empty sites in related phages. Both of these endonucleases cleave intronless DNA polymerase genes at identical positions. This shared feature between an intronic and free-standing endonuclease is unprecedented. Based on this evidence, we propose that introns and their homing endonucleases evolve separately to target the same highly conserved sequences, uniting afterwards to create a composite mobile element.
Project description: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 betabetaalphaalphabeta fold, suggestive of module shuffling between different homing endonuclease families.
Project description:We have recently described three group I introns inserted into a single gene, orf142, of the staphylococcal bacteriophage Twort and suggested the presence of at least two additional self-splicing introns in this phage genome. Here we report that two previously uncharacterized introns, 429 and 1087 nt in length, interrupt the Twort gene coding for the large subunit of ribonucleotide reductase (nrdE). Reverse transcription-polymerase chain reaction (RT-PCR) of RNA isolated from Staphylococcus aureus after phage infection indicates that the introns are removed from the primary transcript in vivo. Both nrdE introns show sequence similarity to the Twort orf142 introns I2 and I3, suggesting either a common origin of these introns or shuffling of intron structural elements. Intron 2 encodes a DNA endonuclease, I-TwoI, with similarity to homing endonucleases of the HNH family. Like I-HmuI and I-HmuII, intron-encoded HNH endonucleases in Bacillus subtilis phages SPO1 and SP82, I-TwoI nicks only one strand of its DNA recognition sequence. However, whereas I-HmuI and I-HmuII cleave the template strand in exon 2, I-TwoI cleaves the coding strand in exon 1. In each case, the 3' OH created on the cut strand is positioned to prime DNA synthesis towards the intron, suggesting that this reaction contributes to the mechanism of intron homing. Both nrdE introns are inserted in highly conserved regions of the ribonucleotide reductase gene, next to codons for functionally important residues.
Project description:Background:There are six known families of homing endonucleases, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which are characterized by their conserved residues. Previously, we discovered a novel homing endonuclease F-CphI encoded by ORF177 of cyanophage S-PM2. F-CphI does not resemble any characterized homing endonucleases. Instead, the C-terminus of F-CphI aligns well with the N-terminal catalytic domain of a Holliday junction DNA resolvase, phage T4 endonuclease VII (Endo VII). Results:A PSI-BLAST search resulted in a total of 313 Endo VII motif-containing sequences in sequenced genomes. Multiple sequence alignment showed that the catalytically important residues of T4 Endo VII were all well conserved in these proteins. Our site-directed mutagenesis studies further confirmed that the catalytically important residues of T4 Endo VII were also essential for F-CphI activity, and thus F-CphI might use a similar protein fold as Endo VII for DNA cleavage. A phylogenetic tree of the Endo VII motif-containing sequences showed that putative resolvases grouped into one clade while putative homing endonucleases and restriction endonucleases grouped into another clade. Conclusions:Based on the unique conserved residues, we proposed that F-CphI represents a new homing endonuclease family, which was named the DHHRN family. Our phylogenetic analysis could be used to predict the functions of many previously unknown proteins.
Project description:HNH endonucleases in bacteriophages play a variety of roles in the phage lifecycle as key components of phage DNA packaging machines. The deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) encodes an HNH endonuclease (GVE2 HNHE). Here, the crystal structure of GVE2 HNHE is reported. This is the first structural study of a thermostable HNH endonuclease from a thermophilic bacteriophage. Structural comparison reveals that GVE2 HNHE possesses a typical ???-metal fold and Zn-finger motif similar to those of HNH endonucleases from other bacteriophages, apart from containing an extra ?-helix, suggesting conservation of these enzymes among bacteriophages. Biochemical analysis suggests that the alanine substitutions of the conserved residues (H93, N109 and H118) in the HNH motif of GVE2 HNHE abolished 94%, 60% and 83% of nicking activity, respectively. Compared to the wild type enzyme, the H93A mutant displayed almost the same conformation while the N108A and H118A mutants had different conformations. In addition, the wild type enzyme was more thermostable than the mutants. In the presence of Mn2+ or Zn2+, the wild type enzyme displayed distinct DNA nicking patterns. However, high Mn2+ concentrations were needed for the N109A and H118A mutants to nick DNA while Zn2+ inactivated their nicking activity.
Project description: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.
Project description:The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5'-GGTAC--C-3' in the presence of Mg2+ as shown generating 3' four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a betabetaalpha-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD...D/EXK superfamily of nucleases, instead is a member of the HNH superfamily.
Project description:Many bacteriophage and prophage genomes encode an HNH endonuclease (HNHE) next to their cohesive end site and terminase genes. The HNH catalytic domain contains the conserved catalytic residues His-Asn-His and a zinc-binding site [CxxC](2). An additional zinc ribbon (ZR) domain with one to two zinc-binding sites ([CxxxxC], [CxxxxH], [CxxxC], [HxxxH], [CxxC] or [CxxH]) is frequently found at the N-terminus or C-terminus of the HNHE or a ZR domain protein (ZRP) located adjacent to the HNHE. We expressed and purified 10 such HNHEs and characterized their cleavage sites. These HNHEs are site-specific and strand-specific nicking endonucleases (NEase or nickase) with 3- to 7-bp specificities. A minimal HNH nicking domain of 76 amino acid residues was identified from Bacillus phage ? HNHE and subsequently fused to a zinc finger protein to generate a chimeric NEase with a new specificity (12-13 bp). The identification of a large pool of previously unknown natural NEases and engineered NEases provides more 'tools' for DNA manipulation and molecular diagnostics. The small modular HNH nicking domain can be used to generate rare NEases applicable to targeted genome editing. In addition, the engineered ZF nickase is useful for evaluation of off-target sites in vitro before performing cell-based gene modification.
Project description:The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel ?-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15-20% amino acid sequence identity. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn²?-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet_0936 may be a DNA repair enzyme.