GIY-YIG Homing Endonuclease Research Articles

Understanding the Evolution of Mitochondrial Genomes in the Green Macroalgal Genus Ulva (Ulvophyceae, Chlorophyta)

To gain more insights into the evolution of mitochondrial genomes (mitogenomes or mtDNAs) in the green macroalgal genus Ulva (Ulvophyceae, Chlorophyta), we sequenced seven Ulva mitogenomes from six species as well as one Percursaria mitogenome as outgroup, and compared them with the available Ulva mtDNA data. Our comparative analyses unveiled many novel findings. First, the Ulva mitogenomes shared a total of 62 core genes including 29 protein-coding genes (PCGs), three ribosomal RNA genes (rRNAs), 26 transfer RNA genes (tRNAs), three conserved free-standing open reading frames (orfs), and one putative RNA subunit of RNase P (rnpB). The rrn5 gene previously unrecognized is present in all sequenced ulvalean mitogenomes, which is situated between trnG(ucc) and trnW(cca). Second, the evolution of tRNAs in Ulva mitogenomes is related to different processes, including duplication, transposition, remolding, degeneration, loss and recruitment of tRNAs. The duplication of three tRNAs, i.e., trnT1(ugu), trnI1(gau), and trnM2(cau), was observed in Ulva mitogenomes. Third, the DNA-directed RNA polymerases (rpos), belonging to single-subunit DNA-dependent RNA polymerase (ssRNAP) family, are common in ulvalean mitogenomes. A total of three full-length and 55 split rpos have been detected in these 33 ulvalean mitogenomes. Fourth, six types of group I/II introns are detected at 29 insertion sites which are related to seven host genes (atp1, cox1, cox2, nad3, nad5, rnl, and rns) in these ulvalean mitogenomes. One group IB intron, i.e., intron cox1-214 which carried a GIY-YIG homing endonuclease (GHE), was observed for the first time in Ulva organelle genomes. Finally, phylogenomic analyses based on mitogenome dataset showed that the Ulva was split into two sister clades, representing Ulva lineage I and II, which was consistent to the results based on plastid genome dataset. Our study provides more important findings to better understand the evolution of mitochondrial genome in green algae.

Mitochondrial Genomics of Six Cacao Pathogens From the Basidiomycete Family Marasmiaceae.

Thread blight disease has recently been described as an emerging disease on cacao (Theobroma cacao) in Ghana. In Ghana, thread blight disease is caused by multiple species of the Marasmiaceae family: Marasmius tenuissimus, M. crinis-equi, M. palmivorus, and Marasmiellus scandens. Interestingly, two additional members of the Marasmiaceae; Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom disease), are major pathogens of cacao in the Western hemisphere. It is important to accurately characterize the genetic relationships among these economically important species in support of their disease management. We used data from Illumina NGS-based genome sequencing efforts to study the mitochondrial genomes (mitogenomes) of the four cacao thread blight associated pathogens from Ghana and compared them with published mitogenomes of Mon. roreri and Mon. perniciosa. There is a remarkable interspecies variation in mitogenome size within the six cacao-associated Marasmiaceae species, ranging from 43,121 to 109,103 bp. The differences in genome lengths are primarily due to the number and lengths of introns, differences in intergenic space, and differences in the size and numbers of unidentified ORFs (uORF). Among seven M. tenuissimus mitogenomes sequenced, there is variation in size and sequence pointing to divergent evolution patterns within the species. The intronic regions show a high degree of sequence variation compared to the conserved sequences of the 14 core genes. The intronic ORFs identified, regardless of species, encode GIY-YIG or LAGLIDADG domain-containing homing endonuclease genes. Phylogenetic relationships using the 14 core proteins largely mimic the phylogenetic relationships observed in gene order patterns, grouping M. tenuissimus with M. crinis-equi, and M. palmivorus with Mon. roreri and Mon. perniciosa, leaving Mar. scandens as an outlier. The results from this study provide evidence of independent expansion/contraction events and sequence diversification in each species and establish a foundation for further exploration of the evolutionary trajectory of the fungi in Marasmiaceae family.

Chloroplast Genomes of the Green-Tide Forming Alga Ulva compressa: Comparative Chloroplast Genomics in the Genus Ulva (Ulvophyceae, Chlorophyta)

To understand the evolution of Ulva chloroplast genomes at intraspecific and interspecific levels, in this study, three complete chloroplast genomes of Ulva compressa Linnaeus were sequenced and compared with the available Ulva cpDNA data. Our comparative analyses unveiled many noticeable findings. First, genome size variations of Ulva cpDNAs at intraspecific and interspecific levels were mainly caused by differences in gain or loss of group I/II introns, integration of foreign DNA fragments, and content of non-coding intergenic spacer regions. Second, chloroplast genomes of U. compressa shared the same 100 conserved genes as other Ulva cpDNA, whereas Ulva flexuosa appears to be the only Ulva species with the minD gene retained in its cpDNA. Third, five types of group I introns, most of which carry a LAGLIDADG or GIY-YIG homing endonuclease, and three of group II introns, usually encoding a reverse transcriptase/maturase, were detected at 26 insertion sites of 14 host genes in the 23 Ulva chloroplast genomes, and many intron insertion-sites have been found for the first time in Chlorophyta. Fourth, one degenerate group II intron previously ignored has been detected in the infA genes of all Ulva species, but not in the closest neighbor, Pseudoneochloris marina, and the other chlorophycean taxa, indicating that it should be the result of an independent invasion event that occurred in a common ancestor of Ulva species. Finally, the seven U. compressa cpDNAs represented a novel gene order which was different from that of other Ulva cpDNAs. The structure of Ulva chloroplast genomes is not conserved, but remarkably plastic, due to multiple rearrangement events.

Annotation and analysis of the mitochondrial genome of Coniothyrium glycines, causal agent of red leaf blotch of soybean, reveals an abundance of homing endonucleases.

Coniothyrium glycines, the causal agent of soybean red leaf blotch, is a USDA APHIS-listed Plant Pathogen Select Agent and potential threat to US agriculture. Sequencing of the C. glycines mt genome revealed a circular 98,533-bp molecule with a mean GC content of 29.01%. It contains twelve of the mitochondrial genes typically involved in oxidative phosphorylation (atp6, cob, cox1-3, nad1-6, and nad4L), one for a ribosomal protein (rps3), four for hypothetical proteins, one for each of the small and large subunit ribosomal RNAs (rns and rnl) and a set of 30 tRNAs. Genes were encoded on both DNA strands with cox1 and cox2 occurring as adjacent genes having no intergenic spacers. Likewise, nad2 and nad3 are adjacent with no intergenic spacers and nad5 is immediately followed by nad4L with an overlap of one base. Thirty-two introns, comprising 54.1% of the total mt genome, were identified within eight protein-coding genes and the rnl. Eighteen of the introns contained putative intronic ORFs with either LAGLIDADG or GIY-YIG homing endonuclease motifs, and an additional eleven introns showed evidence of truncated or degenerate endonuclease motifs. One intron possessed a degenerate N-acetyl-transferase domain. C. glycines shares some conservation of gene order with other members of the Pleosporales, most notably nad6-rnl-atp6 and associated conserved tRNA clusters. Phylogenetic analysis of the twelve shared protein coding genes agrees with commonly accepted fungal taxonomy. C. glycines represents the second largest mt genome from a member of the Pleosporales sequenced to date. This research provides the first genomic information on C. glycines, which may provide targets for rapid diagnostic assays and population studies.

The intron landscape of the mtDNA cytb gene among the Ascomycota: introns and intron-encoded open reading frames

Fungal mitochondrial genes are frequently noted for the presence of introns. These introns are self-splicing and can be assigned to either group I or II introns and they can encode open reading frames (ORFs). This study examines the introns present within the cytochrome b (cytb) gene of ascomycetes fungi. Cytochrome b gene sequences were sampled from GenBank and supplemented with our own data for species of Leptographium and Ophiostoma. Group I introns were encountered most frequently, many encoding either LAGLIDADG or GIY-YIG homing endonucleases (HEs). Numerous examples of different intron/ORF arrangements were observed including nested ORFs, multiple ORFs within a single intron and intron ORFs at various stages of erosion due to the accumulation of mutations. In addition, we noted one example of a nested intron and one complex group II intron that could potentially allow for alternative splicing. Documenting the distribution of introns within the same gene across a range of species allows for a better understanding of the evolution of introns and intronic ORFs. Intron landscapes also are a resource that can help in annotating genes and in bioprospecting for potentially active HEs, which are rare-cutting DNA endonucleases with applications in biotechnology.

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.

Complete mitochondrial genome of the entomopathogenic fungus Beauveria pseudobassiana (Ascomycota, Cordycipitaceae)

The complete mitochondrial genome of the entomopathogenic fungus Beauveria pseudobassiana (Ascomycota, Cordycipitaceae) was determined and found to be 28,006 bp in length. It encodes genes for 14 proteins, 2 ribosomal RNA subunits, 25 transfer RNAs with the synteny identical to those of B. bassiana and B. brongniartii. The overall base composition is 36.7% A, 35.8% T, 15.3% C and 12.2% G with an AT content of 72.5%. Group-І introns were found in large rRNA gene (rnl) and cox2 gene that include rps3 gene and putative GIY-YIG homing endonuclease, respectively. The mitochondrial genome of B. pseudobassiana will contribute to the investigation of the phylogenetic relationship, taxonomic resolution and biogeography of Beauveria.

The monomeric GIY-YIG homing endonuclease I-BmoI uses a molecular anchor and a flexible tether to sequentially nick DNA

The GIY-YIG nuclease domain is found within protein scaffolds that participate in diverse cellular pathways and contains a single active site that hydrolyzes DNA by a one-metal ion mechanism. GIY-YIG homing endonucleases (GIY-HEs) are two-domain proteins with N-terminal GIY-YIG nuclease domains connected to C-terminal DNA-binding and they are thought to function as monomers. Using I-BmoI as a model GIY-HE, we test mechanisms by which the single active site is used to generate a double-strand break. We show that I-BmoI is partially disordered in the absence of substrate, and that the GIY-YIG domain alone has weak affinity for DNA. Significantly, we show that I-BmoI functions as a monomer at all steps of the reaction pathway and does not transiently dimerize or use sequential transesterification reactions to cleave substrate. Our results are consistent with the I-BmoI DNA-binding domain acting as a molecular anchor to tether the GIY-YIG domain to substrate, permitting rotation of the GIY-YIG domain to sequentially nick each DNA strand. These data highlight the mechanistic differences between monomeric GIY-HEs and dimeric or tetrameric GIY-YIG restriction enzymes, and they have implications for the use of the GIY-YIG domain in genome-editing applications.

102 Genome engineering nucleases derived from GIY-YIG homing endonucleases

Efficient targeted manipulation of complex genomes requires highly specific endonucleases to generate double-strand breaks at defined locations (Bibikova et al., 2003; Bogdanove and Voytas, 2011). The predominantly engineered nucleases, zinc-finger nucleases (ZFNs), and TAL effector nucleases (TALENs) use the catalytic domain of FokI as the nuclease portion. This domain, however, functions as a dimer to nonspecifically cleave DNA meaning that ZFNs and TALENs must be designed in head-to-head pairs to target a desired sequence. To overcome this limitation and expand the toolbox of genome editing reagents, we used the N-terminal catalytic domain and interdomain linker of the monomeric GIY-YIG homing endonuclease I-TevI to create I-TevI-zinc-fingers (Tev-ZFEs), and I-TevI-TAL effectors (Tev-TALs) (Kleinstiver et al. 2012). We also made I-TevI fusions to LAGLIDADGs homing endonucleases (I-Tev-LHEs). All the three fusions showed activity on model substrates on par with ZFNs and TALENs in yeast-based recombination assays. These proof-of-concept experiments demonstrate that the catalytic domain of GIY-YIG homing endonucleases can be targeted to relevant loci by fusing the domain to characterize DNA-binding platforms. Recent efforts have focused on improving the Tev-TAL platform by (1) understanding the spacing requirements between the nuclease cleavage site and the DNA binding site, (2) probing the DNA binding requirements of the I-TevI linker domain, and (3) demonstrating activity in mammalian systems.

Monomeric site-specific nucleases for genome editing

Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genome-editing applications. Fusion of the GIY-YIG nuclease domain to three-member zinc-finger DNA binding domains generated chimeric GIY-zinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5'-CNNNG-3' cleavage motif. The Tev-ZFEs function to induce recombination in a yeast-based assay with activity on par with a homodimeric Zif268 zinc-finger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5'-CNNNG-3' motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.

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.

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.

A unified genetic, computational and experimental framework identifies functionally relevant residues of the homing endonuclease I-BmoI.

Insight into protein structure and function is best obtained through a synthesis of experimental, structural and bioinformatic data. Here, we outline a framework that we call MUSE (mutual information, unigenic evolution and structure-guided elucidation), which facilitated the identification of previously unknown residues that are relevant for function of the GIY-YIG homing endonuclease I-BmoI. Our approach synthesizes three types of data: mutual information analyses that identify co-evolving residues within the GIY-YIG catalytic domain; a unigenic evolution strategy that identifies hyper- and hypo-mutable residues of I-BmoI; and interpretation of the unigenic and co-evolution data using a homology model. In particular, we identify novel positions within the GIY-YIG domain as functionally important. Proof-of-principle experiments implicate the non-conserved I71 as functionally relevant, with an I71N mutant accumulating a nicked cleavage intermediate. Moreover, many additional positions within the catalytic, linker and C-terminal domains of I-BmoI were implicated as important for function. Our results represent a platform on which to pursue future studies of I-BmoI and other GIY-YIG-containing proteins, and demonstrate that MUSE can successfully identify novel functionally critical residues that would be ignored in a traditional structure-function analysis within an extensively studied small domain of ∼90 amino acids.

Unconventional GIY-YIG homing endonuclease encoded in group I introns in closely related strains of the Bacillus cereus group

Several group I introns have been previously found in strains of the Bacillus cereus group at three different insertion sites in the nrdE gene of the essential nrdIEF operon coding for ribonucleotide reductase. Here, we identify an uncharacterized group IA intron in the nrdF gene in 12 strains of the B. cereus group and show that the pre-mRNA is efficiently spliced. The Bacillus thuringiensis ssp. pakistani nrdF intron encodes a homing endonuclease, denoted I-BthII, with an unconventional GIY-(X)8-YIG motif that cleaves an intronless nrdF gene 7 nt upstream of the intron insertion site, producing 2-nt 3′ extensions. We also found four additional occurrences of two of the previously reported group I introns in the nrdE gene of 25 sequenced B. thuringiensis and one B. cereus strains, and one non-annotated group I intron at a fourth nrdE insertion site in the B. thuringiensis ssp. Al Hakam sequenced genome. Two strains contain introns in both the nrdE and the nrdF genes. Phylogenetic studies of the nrdIEF operon from 39 strains of the B. cereus group suggest several events of horizontal gene transfer for two of the introns found in this operon.

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.

A Functional Homing Endonuclease in the Bacillus anthracis nrdE Group I Intron

The essential Bacillus anthracis nrdE gene carries a self-splicing group I intron with a putative homing endonuclease belonging to the GIY-YIG family. Here, we show that the nrdE pre-mRNA is spliced and that the homing endonuclease cleaves an intronless nrdE gene 5 nucleotides (nt) upstream of the intron insertion site, producing 2-nt 3' extensions. We also show that the sequence required for efficient cleavage spans at least 4 bp upstream and 31 bp downstream of the cleaved coding strand. The position of the recognition sequence in relation to the cleavage position is as expected for a GIY-YIG homing endonuclease. Interestingly, nrdE genes from several other Bacillaceae were also susceptible to cleavage, with those of Bacillus cereus, Staphylococcus epidermidis (nrdE1), B. anthracis, and Bacillus thuringiensis serovar konkukian being better substrates than those of Bacillus subtilis, Bacillus lichenformis, and S. epidermidis (nrdE2). On the other hand, nrdE genes from Lactococcus lactis, Escherichia coli, Salmonella enterica serovar Typhimurium, and Corynebacterium ammoniagenes were not cleaved. Intervening sequences (IVSs) residing in protein-coding genes are often found in enzymes involved in DNA metabolism, and the ribonucleotide reductase nrdE gene is a frequent target for self-splicing IVSs. A comparison of nrdE genes from seven gram-positive low-G+C bacteria, two bacteriophages, and Nocardia farcinica showed five different insertion sites for self-splicing IVSs within the coding region of the nrdE gene.

Distance determination by GIY-YIG intron endonucleases: discrimination between repression and cleavage functions

GIY-YIG homing endonucleases are modular proteins, with conserved N-terminal catalytic domains connected by linkers to C-terminal DNA-binding domains. I-TevI, the T4 phage GIY-YIG intron endonuclease, functions both in promoting td intron homing, and in acting as a transcriptional autorepressor. Repression is achieved by binding to an operator, which is cleaved at 100-fold reduced efficiency relative to the intronless homing site. The linker includes a zinc finger, which functions in distance determination, to constrain the catalytic domain to cleave the homing site at a fixed position. Here we show that I-BmoI, a related GIY-YIG endonuclease lacking a zinc finger, also possesses some cleavage distance discrimination. Furthermore, hybrid endonucleases constructed by swapping the domains of I-BmoI and I-TevI are active, precise and demonstrate that features other than the zinc finger facilitate distance determination. Most importantly, I-TevI zinc finger mutants cleave the operator more efficiently than the homing site, the converse of wild-type protein. These results are consistent with the zinc finger acting as a measuring device, directing efficient cleavage of the homing site to promote intron mobility, while reducing cleavage at the operator to ensure transcriptional autorepression and phage viability.

Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns.

All genetic markers from phage T2 are partially excluded from the progeny of mixed infections with the related phage T4 (general, or phage exclusion). Several loci, including gene 56 of T2, are more dramatically excluded, being present in only approximately 1% of the progeny. This phenomenon is referred to as localized marker exclusion. Gene 69 is adjacent to gene 56 of T4 but is absent in T2, being replaced by completely nonhomologous DNA. We describe SegF, a novel site-specific DNA endonuclease encoded by gene 69, which is similar to GIY-YIG homing endonucleases of group I introns. Interestingly, SegF preferentially cleaves gene 56 of T2, both in vitro and in vivo, compared with that of phage T4. Repair of the double-strand break (DSB) results in the predominance of T4 genes 56 and segF in the progeny, with exclusion of the corresponding T2 sequences. Localized exclusion of T2 gene 56 is dependent on full-length SegF and is likely analogous to group I intron homing, in which repair of a DSB results in coconversion of markers in the flanking DNA. Phage T4 has many optional homing endonuclease genes similar to segF, whereas similar endonuclease genes are relatively rare in other members of the T-even family of bacteriophages. We propose that the general advantage enjoyed by T4 phage, over almost all of its relatives, is a cumulative effect of many of these localized events.