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• W2086103567 abstract "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 [1Belfort M. Derbyshire V. Parker M.M. Cousineau B. Lambowitz A.M. Mobile introns: pathways and proteins.in: Craig N. DNA Mobile II. ASM Press, Washington, DC2002: 761-783Google Scholar]. 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 ΦI 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. 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 [1Belfort M. Derbyshire V. Parker M.M. Cousineau B. Lambowitz A.M. Mobile introns: pathways and proteins.in: Craig N. DNA Mobile II. ASM Press, Washington, DC2002: 761-783Google Scholar]. 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 ΦI 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. Gene 5.3 is a small open reading frame (ORF) of unknown function located adjacent to the DNA polymerase gene (gene 5) in both T7 and the related phage T3. Despite a lack of similarity to each other, each gene shows similarity to homing endonucleases from different families. T7 gp5.3 shows slight similarity to the cyanobacterial intron endonuclease I-Ssp6803I [2Biniszkiewicz D. Cesnaviciene E. Shub D.A. Self-splicing group I intron in cyanobacterial initiator methionine tRNA: evidence for lateral transfer of introns in bacteria.EMBO J. 1994; 13: 4629-4635Crossref PubMed Scopus (64) Google Scholar, 3Bonocora R.P. Shub D.A. A novel group I intron-encoded endonuclease specific for the anticodon region of tRNA(fMet) genes.Mol. Microbiol. 2001; 39: 1299-1306Crossref PubMed Google Scholar, 4Orlowski J. Boniecki M. Bujnicki J.M. I-Ssp6803I: the first homing endonuclease from the PD-(D/E)XK superfamily exhibits an unusual mode of DNA recognition.Bioinformatics. 2007; 23: 527-530Crossref PubMed Scopus (28) Google Scholar, 5Zhao L. Bonocora R.P. Shub D.A. Stoddard B.L. The restriction fold turns to the dark side: a bacterial homing endonuclease with a PD-(D/E)-XK motif.EMBO J. 2007; 26: 2432-2442Crossref PubMed Scopus (46) Google Scholar], whereas T3 gp5.3 (and homologous ORFs in Yersinia phage ΦYeO3-12 [6Pajunen M.I. Kiljunen S.J. Soderholm M.E. Skurnik M. Complete genomic sequence of the lytic bacteriophage phiYeO3–12 of Yersinia enterocolitica serotype O:3.J. Bacteriol. 2001; 183: 1928-1937Crossref PubMed Scopus (76) Google Scholar] and Pseudomonas phage PA11 [7Kwan T. Liu J. Dubow M. Gros P. Pelletier J. Comparative genomic analysis of 18 Pseudomonas aeruginosa bacteriophages.J. Bacteriol. 2006; 188: 1184-1187Crossref PubMed Scopus (112) Google Scholar]) contains the HNH homing endonuclease motif [8Shub D.A. Goodrich-Blair H. Eddy S.R. Amino acid sequence motif of group I intron endonucleases is conserved in open reading frames of group II introns.Trends Biochem. Sci. 1994; 19: 402-404Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 9Gorbalenya A.E. Self-splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family.Protein Sci. 1994; 3: 1117-1120Crossref PubMed Scopus (101) Google Scholar]. The T7-like phages ΦI, W31, and K1F lack gene 5.3, having just a 19 bp separation between genes 5 and 5.5 that is AT rich and contains the ribosome binding site for gene 5.5. However, these phages have a 601 bp group I intron inserted 156 bp from the end of gene 5 [10Bonocora R.P. Shub D.A. A self-splicing group I intron in DNA polymerase genes of T7-like bacteriophages.J. Bacteriol. 2004; 186: 8153-8155Crossref PubMed Scopus (12) Google Scholar, 11Scholl D. Merril C. The genome of bacteriophage K1F, a T7-like phage that has acquired the ability to replicate on K1 strains of Escherichia coli.J. Bacteriol. 2005; 187: 8499-8503Crossref PubMed Scopus (55) Google Scholar]. These introns encode a 131 codon ORF belonging to the HNH endonuclease gene family, inserted into stem P6a [10Bonocora R.P. Shub D.A. A self-splicing group I intron in DNA polymerase genes of T7-like bacteriophages.J. Bacteriol. 2004; 186: 8153-8155Crossref PubMed Scopus (12) Google Scholar]. Yersinia pestis phage A1122 has neither a gene 5.3 nor a group I intron [12Garcia E. Elliott J.M. Ramanculov E. Chain P.S. Chu M.C. Molineux I.J. The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.J. Bacteriol. 2003; 185: 5248-5262Crossref PubMed Scopus (73) Google Scholar]. PCR analysis of the gene 5.3 locus from five additional phages in this family (ΦIIP, ΦIIW, H, ViIII, and C21R) did not detect any additional insertions (data not shown). Phage ΦIIP, which lacks any insertion in this region, was selected for further study. Schematic representations of this region in the phages used in this study are presented in Figure 1. Database searches with BLASTP [13Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (56908) Google Scholar] with the translated intron-encoded ORF from phage ΦI reveal the next highest similarities to intron-encoded ORFs from W31 and K1F followed by ORFs at the gene 5.3 locus of phages T3, ΦYE03-12, and PA11. These relationships were confirmed by phylogenetic reconstruction with PHYML (not shown). The 101 amino acid T3 gp5.3 aligns over its entire length with the intron-encoded ORFs, with especially strong conservation in the HNH region (Figure S1 available online). This similarity between the intron-encoded ORFs and T3 gp5.3 prompted us to determine the function of these proteins. The in vitro synthesized ΦI and W31 intron-encoded proteins were assayed for endonuclease activity on synthetic DNA substrates corresponding to intronless ΦI and W31 DNA polymerase sequences. Both proteins nick the bottom strand of each target DNA but are not active on the top strands (Figure 2A). This endonuclease has been named I-TslI (Intron-encoded endonuclease from T-seven-like phages, I). Free-standing (non-intron or -intein encoded) ORFs of bacteriophages, eubacteria, and eukaryotes have also been identified that contain homing endonuclease signature motifs [14Lambowitz A.M. Belfort M. Introns as mobile genetic elements.Annu. Rev. Biochem. 1993; 62: 587-622Crossref PubMed Scopus (525) Google Scholar]. Notably, the genome of bacteriophage T4 is infested with ORFs bearing similarity to homing endonucleases [15Miller E.S. Kutter E. Mosig G. Arisaka F. Kunisawa T. Ruger W. Bacteriophage T4 genome.Microbiol. Mol. Biol. Rev. 2003; 67: 86-156Crossref PubMed Scopus (495) Google Scholar]. In addition to the endonuclease genes in its three self-splicing group I introns, T4 has at least 12 homing endonuclease-like ORFs inserted between genes that are conserved in other T-even phages. Several of these encode endonucleases [15Miller E.S. Kutter E. Mosig G. Arisaka F. Kunisawa T. Ruger W. Bacteriophage T4 genome.Microbiol. Mol. Biol. Rev. 2003; 67: 86-156Crossref PubMed Scopus (495) Google Scholar, 16Belle A. Landthaler M. Shub D.A. Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns.Genes Dev. 2002; 16: 351-362Crossref PubMed Scopus (57) Google Scholar, 17Kadyrov F.A. Shlyapnikov M.G. Kryukov V.M. A phage T4 site-specific endonuclease, SegE, is responsible for a non-reciprocal genetic exchange between T-even-related phages.FEBS Lett. 1997; 415: 75-80Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 18Liu Q. Belle A. Shub D.A. Belfort M. Edgell D.R. SegG endonuclease promotes marker exclusion and mediates co-conversion from a distant cleavage site.J. Mol. Biol. 2003; 334: 13-23Crossref PubMed Scopus (33) Google Scholar, 19Sharma M. Ellis R.L. Hinton D.M. Identification of a family of bacteriophage T4 genes encoding proteins similar to those present in group I introns of fungi and phage.Proc. Natl. Acad. Sci. USA. 1992; 89: 6658-6662Crossref PubMed Scopus (75) Google Scholar, 20Shcherbakov V. Granovsky I. Plugina L. Shcherbakova T. Sizova S. Pyatkov K. Shlyapnikov M. Shubina O. Focused genetic recombination of bacteriophage t4 initiated by double-strand breaks.Genetics. 2002; 162: 543-556PubMed Google Scholar, 21Brok-Volchanskaya V.S. Kadyrov F.A. Sivogrivov D.E. Kolosov P.M. Sokolov A.S. Shlyapnikov M.G. Kryukov V.M. Granovsky I.E. Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.Nucleic Acids Res. 2008; 36: 2094-2105Crossref PubMed Scopus (24) Google Scholar] that are themselves mobile, invading vacant sites in other phages by a gene conversion mechanism analogous to intron homing [16Belle A. Landthaler M. Shub D.A. Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns.Genes Dev. 2002; 16: 351-362Crossref PubMed Scopus (57) Google Scholar, 17Kadyrov F.A. Shlyapnikov M.G. Kryukov V.M. A phage T4 site-specific endonuclease, SegE, is responsible for a non-reciprocal genetic exchange between T-even-related phages.FEBS Lett. 1997; 415: 75-80Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 18Liu Q. Belle A. Shub D.A. Belfort M. Edgell D.R. SegG endonuclease promotes marker exclusion and mediates co-conversion from a distant cleavage site.J. Mol. Biol. 2003; 334: 13-23Crossref PubMed Scopus (33) Google Scholar, 21Brok-Volchanskaya V.S. Kadyrov F.A. Sivogrivov D.E. Kolosov P.M. Sokolov A.S. Shlyapnikov M.G. Kryukov V.M. Granovsky I.E. Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.Nucleic Acids Res. 2008; 36: 2094-2105Crossref PubMed Scopus (24) Google Scholar, 22Sandegren L. Nord D. Sjoberg B.M. SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages.Nucleic Acids Res. 2005; 33: 6203-6213Crossref PubMed Scopus (26) Google Scholar], a process called “intronless homing” [16Belle A. Landthaler M. Shub D.A. Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns.Genes Dev. 2002; 16: 351-362Crossref PubMed Scopus (57) Google Scholar]. In vitro protein synthesis of T3 or T7 gene 5.3 gave poor expression yields and endonuclease activity could not be demonstrated (data not shown). Therefore, we assayed phage-infected cell extracts for endonuclease activity with PCR-amplified ΦIIP substrate DNA. No activity was observed from ΦIIP or T7 extracts (data not shown). However, both ΦI and T3 extracts demonstrated cleavage activity, with optimal activity appearing 10 min postinfection (Figure 2B). Cleavage of top strands was not detected (data not shown). Deletions that remove most of the coding sequences of I-TslI and gp5.3 eliminate activity (Figure 2B). These results indicate that I-TslI and T3 gp5.3 are transcribed and translated during phage infection and that the endonuclease activity in T3 extracts is due, at least in part, to gp5.3. However, because we were unable to synthesize active protein in vitro, it remains to be determined whether gp5.3 is sufficient for the activity. After the convention suggested for free-standing homing endonucleases, the activity of gp5.3 is designated F-TslI [23Roberts R.J. Belfort M. Bestor T. Bhagwat A.S. Bickle T.A. Bitinaite J. Blumenthal R.M. Degtyarev S. Dryden D.T. Dybvig K. et al.A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes.Nucleic Acids Res. 2003; 31: 1805-1812Crossref PubMed Scopus (501) Google Scholar]. The DNA polymerase genes from the T7-like phages described in this study provide a sampling of natural target site variants for I-TslI and F-TslI. We tested extracts from cells infected with phages ΦI, ΦIΔTsl (lacking 73 of 131 residues of I-TslI, including the HNH catalytic domain), T3, and T3Δ5.3 (lacking 94 of 101 residues of F-TslI) on DNA substrates amplified from phage ΦIIP, T3, T7, and ΦI, and also on double-stranded oligonucleotides representing fused exon sequences from both ΦI and W31. I-TslI and F-TslI cleave the ΦIIP, T7, and intronless ΦI and W31 substrates, but are inactive on a substrate containing the ΦI intron (Figures 2C and 2D). Interestingly, the T3 DNA polymerase sequence is sensitive to cleavage by I-TslI but resistant to cleavage by T3-encoded F-TslI. A summary of the cleavage results is shown in Figure 3B. The exact positions of cleavage by I-TslI and F-TslI were determined on the T7 target sequence. In vitro synthesized I-TslI and extracts from ΦI- and T3-infected cells cut the template strand of the T7 DNA polymerase gene at the same position, one nucleotide 5′ of the IIS (Figure 3A). A portion of the I-TslI cleavage reaction (lane 1) was mixed with the C and T sequencing reactions to eliminate any discrepancy in migration (Figure 3A, lanes C+ and T+, respectively). An extra band corresponding to the cleavage product is apparent in the C+ lane, whereas no extra band can be seen in the T+ lane, indicating that the cleavage product comigrates with the T band. To investigate homing of the ΦI intron, we constructed an isogenic derivative, ΦIΔI, with an exact deletion of the intron in its DNA polymerase gene. After coinfection of ΦIΔI and ΦI, the fraction of intron-containing individuals increased from an average of 54% of the input phages to ∼92% of the progeny (Table 1). Preferential inheritance of the intron is dependent on integrity of I-TslI, because it did not occur in coinfection between ΦIΔI and ΦIΔTsl.Table 1Endonuclease I-TslI-Dependent HomingCrossExpMOIInput I+ aOligonucleotide ΦId2 was used to identify the presence of the intron.Progeny I+ aOligonucleotide ΦId2 was used to identify the presence of the intron.ΦI×ΦIΔI190.650.922300.440.943250.520.88ΦIΔTslI×ΦIΔI170.640.472180.460.413180.480.39Abbreviations: MOI, multiplicity of infectiona Oligonucleotide ΦId2 was used to identify the presence of the intron. Open table in a new tab Abbreviations: MOI, multiplicity of infection Phage T3 is not compatible with most other T7-like phages in mixed infections [24Hausmann R. The T7 group.in: Calendar R. The Bacteriophages. Plenum Press, New York1988Google Scholar]. To investigate homing directed by F-TslI, we constructed a T3 variant whose sequence at the cleavage site in gene 5 is sensitive to its own endonuclease. The fused exon sequence of ΦIΔI, which contains nine base pair differences from the T3 sequence near the cleavage site, is cleaved by F-TslI (Figures 2D and 3B). We incorporated all nine of these substitutions in T3Δ5.3 to create a sensitive cleavage site (CSS) in a T3 genetic background. In mixed infections, both the wild-type cleavage site (CSR) and the intact gene 5.3 were preferentially inherited in an F-TslI-dependent manner (Table 2). Interestingly, a small fraction of the progeny contains the CSR allele from the donor and the Δ5.3 allele from the recipient, illustrating that recombination is readily detected between these closely spaced loci. The reciprocal recombinant, uniting CSS and intact gene 5.3, should be lethal and was not detected.Table 2Endonuclease F-TslI-Dependent HomingCrossExpMOIInputProgenyCSR aOligonucleotide T3IIS was used to identify the cleavage-resistant wild-type (CSR) T3 sequence.gene5.3bOligonucleotide T35.3 was used to identify intact T3 gene 5.3.CSR aOligonucleotide T3IIS was used to identify the cleavage-resistant wild-type (CSR) T3 sequence.gene5.3bOligonucleotide T35.3 was used to identify intact T3 gene 5.3.T3/CSR×T3Δ5.3/CSS1160.570.570.910.892290.610.610.950.943350.570.570.920.92T3Δ5.3×T3Δ5.3/CSS1170.42NA0.49NA2330.47NA0.48NA3350.47NA0.45NAAbbreviations: MOI, multiplicity of infection; NA, not applicablea Oligonucleotide T3IIS was used to identify the cleavage-resistant wild-type (CSR) T3 sequence.b Oligonucleotide T35.3 was used to identify intact T3 gene 5.3. Open table in a new tab Abbreviations: MOI, multiplicity of infection; NA, not applicable The self-splicing group I intron in ΦI, W31, and K1F interrupts a conserved, functionally important region of the phage-encoded DNA polymerase gene [10Bonocora R.P. Shub D.A. A self-splicing group I intron in DNA polymerase genes of T7-like bacteriophages.J. Bacteriol. 2004; 186: 8153-8155Crossref PubMed Scopus (12) Google Scholar], being inserted immediately adjacent to the aspartate residue that coordinates Mg2+ in the enzyme catalytic center (Figure S2). We have shown that this intron encodes I-TslI, an HNH-like homing endonuclease (Figure 2, Figure 3). It is intriguing that the closest relatives to I-TslI are the three genes 5.3 from phages PA11, Ye03-12, and T3, which also encode an endonuclease, F-TslI (Figure 2; Figure S1). In addition to sequence similarity, I-TslI and F-TslI share functional characteristics. Both enzymes generate a single-strand nick at precisely the same position of the template DNA strand, one nucleotide 5′ of the IIS (Figure 3A) and promote homing reactions in vivo (Table 1, Table 2). Prevention of self-cleavage is a hallmark of homing, whether it is mediated by an intron/intein-encoded or by a free-standing endonuclease. For intron and intein homing, protection from endonuclease cleavage is generally a consequence of the DNA binding site and/or the cleavage site being split by insertion of the respective intervening sequence [25Belfort M. Reaban M.E. Coetzee T. Dalgaard J.Z. Prokaryotic introns and inteins: a panoply of form and function.J. Bacteriol. 1995; 177: 3897-3903Crossref PubMed Google Scholar]. However, in intronless homing the target site is not afforded this protection. Instead, the endonuclease distinguishes self from non-self by using sequence polymorphisms in the DNA target [16Belle A. Landthaler M. Shub D.A. Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns.Genes Dev. 2002; 16: 351-362Crossref PubMed Scopus (57) Google Scholar, 18Liu Q. Belle A. Shub D.A. Belfort M. Edgell D.R. SegG endonuclease promotes marker exclusion and mediates co-conversion from a distant cleavage site.J. Mol. Biol. 2003; 334: 13-23Crossref PubMed Scopus (33) Google Scholar]. In this respect, I-TslI and F-TslI act as typical homing endonucleases as neither enzyme cleaves its own DNA under the same conditions where the target sequence of related phages is cleaved extensively (Figure 2). T3 gene 5 has unique sequence polymorphisms in the highly conserved DNA target region (Figure 3B), one or more of which may enable F-TslI to discriminate self from non-self in DNA binding and/or cleavage reactions. Interestingly, F-TslI is also unable to cleave the intron-containing target (Figure 2C) implying that the intron lies within the cleavage/recognition sites for both I-TslI and F-TslI. It has been proposed that homing endonuclease genes are selfish DNAs that have invaded splicing elements. This association is thought to be mutually advantageous, offering a phenotypically silent home for the endonuclease gene while providing mobility to the splicing element [25Belfort M. Reaban M.E. Coetzee T. Dalgaard J.Z. Prokaryotic introns and inteins: a panoply of form and function.J. Bacteriol. 1995; 177: 3897-3903Crossref PubMed Google Scholar]. Multiple lines of evidence support this hypothesis [26Lambowitz A.M. Infectious introns.Cell. 1989; 56: 323-326Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 27Belfort M. Bacteriophage introns: parasites within parasites?.Trends Genet. 1989; 5: 209-213Abstract Full Text PDF PubMed Scopus (33) Google Scholar, 28Mota E.M. Collins R.A. Independent evolution of structural and coding regions in a Neurospora mitochondrial intron.Nature. 1988; 332: 654-656Crossref PubMed Scopus (60) Google Scholar]. Although most homing endonucleases apparently confer no selective advantage on their host genomes and could be quickly eliminated by mutation, homing allows their propagation and maintenance in the population [29Goddard M.R. Burt A. Recurrent invasion and extinction of a selfish gene.Proc. Natl. Acad. Sci. USA. 1999; 96: 13880-13885Crossref PubMed Scopus (267) Google Scholar]. Although free-standing endonuclease genes are usually restricted to intergenic, nonessential locations, endonuclease invasion of an intron (or intein) greatly expands their potential insertion sites to include essential coding sequences. The most interesting feature shared by endonucleases I-TslI and F-TslI is their ability to cleave DNA at the identical position within the DNA polymerase gene (Figure 3A). This feature, along with the similarity of their sequences, strongly suggests that they have a recent common ancestor. In addition, the close proximity of insertion sites of the genes encoding these endonucleases, only 156 bp apart in the genomes of closely related phages, is highly provocative. Either I-TslI escaped from its intron home into the adjacent intercistronic space, or F-TslI jumped from its intercistronic position into a nearby intron. As we will explain below, we find the latter to be the more likely sequence of events. This scenario could help us to understand the origin of group I intron homing. In order for an endonuclease to be maintained in its new environment after being inserted into an intron, it must be able to promote the spread of the intron by recognizing and cleaving intronless alleles. Two alternative scenarios, both of which require rare events, were considered by Loizos et al. [30Loizos N. Tillier E.R. Belfort M. Evolution of mobile group I introns: recognition of intron sequences by an intron-encoded endonuclease.Proc. Natl. Acad. Sci. USA. 1994; 91: 11983-11987Crossref PubMed Scopus (58) Google Scholar] as explanations of how this may have occurred. One proposal was suggested by the observation that the IIS of the sunY/nrdB intron resembles short sequences flanking its homing endonuclease gene, I-TevII. Moreover, when these flanking sequences were fused, the resultant sequence could be cleaved by the enzyme. They suggested that the endonuclease gene had originally evolved to cut a closely related sequence and was inserted into the intron as a consequence of repair of a cleavage event at this ectopic site. Because there would not necessarily be specificity for the IIS at this new location, survival of the endonuclease would depend on transposition of the intron to a new genomic location, presumably the site for which the enzyme was already adapted. However, this pathway will produce functional gene products only if there is sufficient sequence similarity between the donor and recipient DNA to initiate replicative repair, if the intron is capable of splicing in its new environment, and if the changes brought about by coconversion of flanking DNA are compatible with the function of the final gene product. These requirements are especially unlikely to be met at locations where many group I introns reside: coding sequences of highly conserved enzyme active centers. In a second scenario, considered by Loizos et al. to be less likely [30Loizos N. Tillier E.R. Belfort M. Evolution of mobile group I introns: recognition of intron sequences by an intron-encoded endonuclease.Proc. Natl. Acad. Sci. USA. 1994; 91: 11983-11987Crossref PubMed Scopus (58) Google Scholar], a rare, nonhomologous recombination event could have inserted an endonuclease, which already had specificity for the IIS, into the intron. We suggest that the intron in phages ΦI, W31, and K1F acquired its endonuclease gene by this route, and that this may be a general pathway for the origin of these composite mobile elements. Group I introns and inteins tend to be inserted into highly conserved sequences encoding functionally significant regions of essential genes [31Edgell D.R. Shub D.A. Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites.Proc. Natl. Acad. Sci. USA. 2001; 98: 7898-7903Crossref PubMed Scopus (43) Google Scholar, 32Landthaler M. Begley U. Lau N.C. Shub D.A. Two self-splicing group I introns in the ribonucleotide reductase large subunit gene of Staphylococcus aureus phage Twort.Nucleic Acids Res. 2002; 30: 1935-1943Crossref PubMed Scopus (46) Google Scholar, 33Edgell D.R. Belfort M. Shub D.A. Barriers to intron promiscuity in bacteria.J. Bacteriol. 2000; 182: 5281-5289Crossref PubMed Scopus (115) Google Scholar, 34Derbyshire V. Belfort M. Lightning strikes twice: intron-intein coincidence.Proc. Natl. Acad. Sci. USA. 1998; 95: 1356-1357Crossref PubMed Scopus (34) Google Scholar, 35Pietrokovski S. Intein spread and extinction in evolution.Trends Genet. 2001; 17: 465-472Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 36Gogarten J.P. Senejani A.G. Zhaxybayeva O. Olendzenski L. Hilario E. Inteins: structure, function, and evolution.Annu. Rev. Microbiol. 2002; 56: 263-287Crossref PubMed Scopus (160) Google Scholar, 37Goodwin T.J. Butler M.I. Poulter R.T. Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes.BMC Biol. 2006; 4: 38Crossref PubMed Scopus (14) Google Scholar]. The only way a gene can rid itself of an intron in such a position is by exact deletion. Inexact deletion would leave a lethal insertion/deletion or frame shift [33Edgell D.R. Belfort M. Shub D.A. Barriers to intron promiscuity in bacteria.J. Bacteriol. 2000; 182: 5281-5289Crossref PubMed Scopus (115) Google Scholar, 36Gogarten J.P. Senejani A.G. Zhaxybayeva O. Olendzenski L. Hilario E. Inteins: structure, function, and evolution.Annu. Rev. Microbiol. 2002; 56: 263-287Crossref PubMed Scopus (160) Google Scholar]. We assume that prior to acquiring homing endonuclease genes, group I introns were inserted randomly but survived only where they could not be easily eliminated. Conversely, successful elimination of introns from nonessential coding regions may be responsible for some of the variability in contemporary genomes. On the other hand, for free-standing endonuclease genes to survive in their intercistronic locations, they must be propagated by intronless homing, with target sequences in an adjacent gene [16Belle A. Landthaler M. Shub D.A. Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns.Genes Dev. 2002; 16: 351-362Crossref PubMed Scopus (57) Google Scholar, 18Liu Q. Belle A. Shub D.A. Belfort M. Edgell D.R. SegG endonuclease promotes marker exclusion and mediates co-conversion from a distant cleavage site.J. Mol. Biol. 2003; 334: 13-23Crossref PubMed Scopus (33) Google Scholar]. Functionally important regions of essential genes provide optimal targets, because these will be most frequently encountered and not easily changed. Because optimal IISs and endonuclease cleavage sites share the same properties, introns and endonuclease genes are likely to converge independently on the same set of almost universally conserved sequences. By residing within an intron, an endonuclease can transpose to new sites within coding sequences without necessarily inactivating them and, having acquired an endonuclease that is preadapted for the IIS, the intron is immediately able to increase its frequency in a population by homing. Thus, when an intron and an endonuclease happen to converge on the same conserved DNA target, their combination into a single genetic element should provide substantial selective advantage to both of them, assuring recovery of rare recombination events. Figure 4 illustrates how an intron and an endonuclease gene could have come together in the DNA polymerase gene of T7-like phages. An ancestral phage (A), such as ΦIIP, acquires an intron, perhaps by reverse splicing into mRNA followed by reverse transcription [38Roman J. Woodson S.A. Reverse splicing of the Tetrahymena IVS: evidence for multiple reaction sites in the 23S rRNA.RNA. 1995; 1: 478-490PubMed Google Scholar, 39Roman J. Woodson S.A. Integration of the Tetrahymena group I intron into bacterial rRNA by reverse splicing in vivo.Proc. Natl. Acad. Sci. USA. 1998; 95: 2134-2139Crossref PubMed Scopus (58) Google Scholar], to create phage B. The intron is stably inserted in this site, but cannot spread through the population because of a lack of a homing mechanism. Other ancestral phages independently acquire endonuclease F-TslI by insertion into the intercistronic region between genes 5 and 5.5. Presumably cleavage activity is initially weak, and this phage (C) is under selection to alter the target sequence in its gene 5, mostly by third position changes, creating a phage similar to phage T3. Simultaneously, to facilitate its spread through the population by homing, the endonuclease is under selection for more efficient cleavage of the unaltered target sequence. Recombination in T7-like phages is very efficient [40Studier F.W. The genetics and physiology of bacteriophage T7.Virology. 1969; 39: 562-574Crossref PubMed Scopus (310) Google Scholar], so the eventual encounter of phages B and C in a mixed infection will lead to creation of the recombinant phage (D). This combination is stable and should spread through the population by homing to intronless sites, with very efficient coconversion of the intron and nearby endonuclease gene. Because it requires the participation of two independent genetic units, we have named this process “collaborative homing” to distinguish it from conventional intron homing [41Zeng Q. Bonocora R.P. Shub D.A. A free-standing homing endonuclease targets an intron insertion site in the psbA gene of cyanophages.Curr. Biol. 2009; 19 (this issue): 218-222Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar]. However, a significant proportion of recombinational events will separate the intron and endonuclease gene, similar to what has been shown here for F-TslI and its cleavage resistant site (Table 2) and elsewhere for the T4-encoded free-standing endonuclease SegG [18Liu Q. Belle A. Shub D.A. Belfort M. Edgell D.R. SegG endonuclease promotes marker exclusion and mediates co-conversion from a distant cleavage site.J. Mol. Biol. 2003; 334: 13-23Crossref PubMed Scopus (33) Google Scholar]. A rare DNA rearrangement can result in insertion of the endonuclease gene into the intron (E). This coupling is the most stable, optimal arrangement for both entities. It ensures that the mobility element (the endonuclease) and the protective element (the intron) can never be separated. The chimeric mobile element can spread to intronless sites via homing and, occasionally, transpose to nonallelic coding sequences. Finally, even if eliminated by exact deletion, this recreates an IIS that is subject to reinvasion in a subsequent homing event. Two examples of the arrangement proposed in Figure 4D, where the intron and endonuclease have come together in the same chromosome but where the endonuclease gene has not yet been transferred into the intron, have recently been identified. Aeromonas salmonicida phage 25, which is related to E. coli phage T4, has a group I intron inserted into the gene for a DNA polymerase subunit [42Petrov V.M. Nolan J.M. Bertrand C. Levy D. Desplats C. Krisch H.M. Karam J.D. Plasticity of the gene functions for DNA replication in the T4-like phages.J. Mol. Biol. 2006; 361: 46-68Crossref PubMed Scopus (64) Google Scholar]. Adjacent to the DNA polymerase coding sequence is a gene encoding an endonuclease of the GIY-YIG family that cleaves the homologous intronless sequence of phage T4 (V. Petrov and J.D. Karam, personal communication). Similarly, we have shown that a free-standing homing endonuclease gene is adjacent to the intron-containing psbA gene in cyanobacterial phage S-PM2. The endonuclease (F-CphI) cannot cut intron-containing DNA, but it does cut both the in vitro synthesized IIS of S-PM2 and intronless psbA genes of related phages [41Zeng Q. Bonocora R.P. Shub D.A. A free-standing homing endonuclease targets an intron insertion site in the psbA gene of cyanophages.Curr. Biol. 2009; 19 (this issue): 218-222Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar]. We propose a model whereby group I introns and homing endonuclease genes individually target the same set of highly conserved DNA sequences for insertion and cleavage, respectively, ultimately uniting to form a chimeric mobile element. The same opportunities for the confluence of homing endonuclease genes and group I introns by genetic recombination that we propose here also apply to other genomes (eubacteria, fungal mitochondria, chloroplasts, nuclei of unicellular eukaryotes) where mobile group I introns are frequently found. Because inteins and group I introns have been found as insertions in the same highly conserved DNA targets [32Landthaler M. Begley U. Lau N.C. Shub D.A. Two self-splicing group I introns in the ribonucleotide reductase large subunit gene of Staphylococcus aureus phage Twort.Nucleic Acids Res. 2002; 30: 1935-1943Crossref PubMed Scopus (46) Google Scholar, 34Derbyshire V. Belfort M. Lightning strikes twice: intron-intein coincidence.Proc. Natl. Acad. Sci. USA. 1998; 95: 1356-1357Crossref PubMed Scopus (34) Google Scholar], this process may also apply to the origin of mobile inteins." @default.
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