• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Jun 15, 2003; 31(12): 3071–3077.
PMCID: PMC162337

The nicking homing endonuclease I-BasI is encoded by a group I intron in the DNA polymerase gene of the Bacillus thuringiensis phage Bastille

Abstract

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 ββααβ fold, suggestive of module shuffling between different homing endonuclease families.

INTRODUCTION

Group I introns are found in protein- and RNA-coding genes of a diverse set of organisms. In eukaryotes, group I introns exist in nuclear rRNA genes of fungi and ciliates and in organellar genes of plants, fungi and flagellates (1). They have also been found in cyanobacteria, proteobacteria, Gram-positive bacteria and bacteriophages (2).

A large number of group I introns encode site-specific DNA endonucleases (1). These intron-encoded endonucleases (homing endonucleases) function primarily in a biological process whereby a group I intron is inserted into an intronless version of a gene, at the same position it already occupies in the intron containing homolog, by a duplicative and unidirectional transfer.

Homing endonucleases belong to four families based on the presence of well-conserved sequence motifs that are denoted LAGLIDADG, His-Cys box, GIY-YIG and H-N-H, respectively (3). Most homing endonucleases bind DNA in a sequence-specific fashion, recognizing large stretches of the intron-minus version of the gene in which they are inserted. In general, the recognition site comprises sequences of both exons and the endonucleases generate a double-strand break close to the intron insertion site (4).

Two of the families, the GIY-YIG and H-N-H homing endonucleases, have been found in group I introns, and also as freestanding open-reading frames, in bacteria and their phages (2,5,6). The coliphage T4 td intron-encoded endonuclease I-TevI is the best-studied member of the GIY-YIG family. It is a bipartite enzyme with distinct catalytic and DNA-binding domains (79) that recognizes a 37-bp region in a sequence tolerant manner (10). Members of the H-N-H family are less well understood, as they possess biochemical activities usually not associated with other homing endonucleases. For instance, I-TevIII of phage RB3 makes a double-strand break similar to other endonucleases but is the only homing endonuclease that generates a 5′ overhang instead of the typical 3′ extension (11). The H-N-H endonucleases I-HmuI and I-HmuII, encoded by group I introns in the DNA polymerase genes of Bacillus phages SPO1 and SP82, respectively, are distinct from other intron-encoded endonucleases in that they cleave intron-plus as well as intron-minus alleles, and cut only one strand of their DNA substrate (12). Another example of a nicking H-N-H endonuclease, I-TwoI, is encoded by a group I intron in the ribonucleotide reductase gene of staphylococcal phage Twort (13).

In this work we describe a group I intron in the DNA polymerase gene of Bacillus thuringiensis phage Bastille. Like the SPO1 and SP82 introns, the Bastille intron encodes a nicking DNA endonuclease and its cleavage site is identical to that of the SPO1-encoded enzyme I-HmuI. However, in contrast to I-HmuI, I-BasI has DNA substrate specificity for intron-minus alleles, which is characteristic of a typical homing endonuclease. Sequence comparisons of the C-terminal portion of phage H-N-H endonucleases against the protein database indicate the presence of a conserved sequence motif that has also been found in endonucleases of the GIY-YIG family, likely forming a small DNA-binding module with a globular ββααβ fold.

MATERIALS AND METHODS

Bacterial and bacteriophage strains

Phage Bastille (HER211) and its host B.thuringiensis (HER1211) were obtained from the Felix d’Herelle Reference Center. Escherichia coli XL-1 Blue (Stratagene) was used as the recipient strain for high-frequency plasmid electroporation. Escherichia coli BL21(DE3) pLysE was used as the bacterial host for protein expression.

Plasmids

The plasmid (pBET) for over-expression of I-BasI was generated by PCR amplification of Bastille DNA using primers BIorf-U2 and BIorf-D. The PCR product was digested with NdeI and ligated into the NdeI site of pAii17 (14).

Expression and preparation of I-BasI protein extracts

Cells were grown in LB supplemented with ampicillin (50 µg/ml) at 37°C to A600 = 0.6 and expression was induced by addition of IPTG to a final concentration of 1 mM. Incubation was continued at 37°C for 3 h. Cells were harvested by centrifugation at 6000 g for 20 min, and resuspended in ice-cold 50 mM Tris–HCl (pH 7.2), 1 mM EDTA, 1 mM PMSF, 2 μg/ml leupeptin, 200 mM KCl at a concentration of 6 ml/g cells. The resuspended cells were sonicated to complete lysis and centrifuged at 12 000 g for 1 h. The protein was present in the pellet fraction. The pellet was washed with chilled deionized H2O, resuspended in 1 ml/g (cells) 6 M guanidine hydrochloride, renatured by dialyzing twice against 100× vol of 50 mM potassium phosphate (pH 7.2), 100 mM NaCl, 1 mM DTT and stored in 10% glycerol at –80°C.

Endonuclease assay with extracts from cells expressing I-BasI

PCR-generated DNA fragments, radio-labeled at their 5′ termini, were used as substrates. The intron-minus Bastille DNA substrate was generated by reverse transcription of Bastille RNA isolated 10 min post-infection with primer S2373 and subsequent PCR amplification with primers S2325 and S2373. The intron-plus Bastille DNA substrate was PCR-generated by amplification of Bastille DNA with primers S2327 and S2373. The intron-minus SPO1 DNA substrate was generated by PCR amplification of plasmid pHGO1ΔI with primers 1 and 2 of Goodrich-Blair and Shub (12). Labeled PCR products were purified with QIAquick spin PCR purification. Reactions were performed with 5 × 103 to 3 × 104 c.p.m. of labeled DNA substrate in a 5 µl reaction volume in 50 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 100 mM NaCl, 1mM DTT with 2 µl protein extract. The reactions were allowed to proceed for 10 min at 30°C and terminated by the addition of 2 µl 95% formamide, 20 mM EDTA, 0.05% bromphenol blue and 0.05% xylene cyanol. Reaction products were separated on a 5% denaturing polyacrylamide gel and visualized by autoradiography.

Isolation of Bastille RNA

Bacillus thuringiensis cells were grown at 37°C to an OD540 of 0.4 (~5 × 107 cells/ml) and infected with ~5 phages/cell. Cells (25 ml) were harvested by centrifugation at 5000 g for 10 min at 4°C and washed twice with 10 mM Tris–HCl (pH 7.5 at 4°C), 100 µg/ml chloramphenicol. Cells were resuspended in 100 µl 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 50 µg/ml lysozyme (Sigma) and incubated for 5 min at room temperature. RNA was isolated with the RNeasy Kit (QIAGEN) as explained in the manufacture’s protocol for isolation of total RNA from bacteria. In vitro labeling of RNA with [α-32P]GTP was according to Reinhold-Hurek and Shub (15).

Isolation of Bastille phage DNA

Bacillus thuringiensis cells were grown to an OD540 of ~0.4, infected at a multiplicity of ~0.1 phage/cell, and incubation was continued until lysis was complete. Phages were precipitated in 10% PEG 8000 and phage particles were further purified by centrifugation through a CsCl step gradient according to the bacteriophage λ purification protocol in Sambrook et al. (16). After removal of CsCl by dialysis against 50 mM Tris–HCl (pH 8.0) and 1 mM EDTA, the phage DNA was extracted with phenol.

Bastille DNA southern hybridization

Restriction enzyme digests of Bastille DNA were separated on a 1% agarose gel and vacuum blotted onto a positively charged nylon membrane (Hybond-N+, Amersham) by alkaline transfer. The Bastille DNA blot was probed with radiolabeled phage Twort orf142 intron DNA (17). The probe was generated by random primed labeling of a 992-bp PCR product, derived from amplification of Twort DNA using primers 4 and 5 from Landthaler and Shub (17). Hybridization was carried out in 6× SSC, 5× Denhardt’s solution, 0.1% SDS, 200 µg/ml herring sperm DNA. Four washes at 50°C in 2× SSC, 0.1% SDS were followed by a single wash in 0.2 × SSC, 0.1% SDS at 50°C.

Mapping of I-BasI cleavage sites

Intron-minus Bastille and SPO1 DNA substrates were generated as described above with 32P-labeled bottom strand primers. Labeled PCR products were incubated with 1/10 vol of I-BasI protein extract in 50 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 100 mM NaCl, 1 mM DTT, phenol extracted and separated on a denaturing polyacrylamide gel. Sequence ladder was generated by a cycle sequencing reaction with the end-labeled primer used to make the DNA substrate.

Oligonucleotides

BIorf-U2, 5′-TGGAGGTACCATATGTTTCAAGAAGAG (868–894); BIorf-D, 5′-TGTGGTGCATATGTTATTTTTTACTTAC (complement: 1432–1459); S2325, 5′-GAGAATTACCCAGAACA (510–526); S2327, 5′-TACACCACAT TACTAGA (1451–1467); S2373, 5′-CTAATGCCATTACACGGGAC (complement: 1668–1687). The underlined sequences indicate introduced NdeI restriction sites.

RESULTS

The discovery of a self-splicing group I intron in the thymidylate synthase gene of Bacillus phage β22 (18) prompted us to screen other, morphologically identical phages for the presence of group I introns by in vitro labeling of RNA from infected cells with [α-32P]GTP. Since the 3′ OH of GTP is the nucleophile in the first transesterification step of group I splicing, the GTP will be added at the 5′ end of the excised introns. Using this method, we have described previously the presence of at least five group I introns in the Staphylococcus phage Twort (13,17). Another phage included in this screen was Bastille, infecting B.thuringiensis. GTP-labeling of RNA, isolated from B.thuringiensis 10 or 20 min after infection with Bastille, resulted in an end-labeled RNA product of ~850 nt (Fig. (Fig.1)1) indicating the presence of a self-splicing group I intron in this phage genome.

Figure 1
GTP-labeling of Bastille RNA. Five micrograms of RNA, isolated from B.thuringiensis before (0) and at times indicated after infection with Bastille, was deproteinized and incubated with [α-32P]GTP under self- splicing conditions. ...

In an attempt to clone the Bastille intron, a DNA fragment containing the three Twort orf142 introns (17) was used as a probe for Southern hybridization of restriction enzyme digested Bastille DNA. Two hybridizing fragments (0.7 and 1.0 kb), identified from NsiI-digested DNA, were isolated from a genomic plasmid mini-library and sequenced on both strands. The junction at the NsiI site was confirmed by sequencing this region on the I-BasI expression plasmid, pBET. Analysis of the sequence (GenBank accession no. AY256517) revealed the presence of a group I intron 853 nt in length (Supplementary Material, Fig. S1). A BLAST search of the protein database (19) showed that the interrupted coding sequence was highly similar to DNA polymerase genes of Bacillus phages SPO1 and SP82, which are also interrupted by self-splicing group I introns. Amino acid sequence alignment of the DNA polymerases of these phages further showed that the introns in Bastille, SPO1 and SP82 are inserted at the homologous site (Fig. (Fig.2).2). Amino acids at the site of intron insertion, corresponding to a region of the palm subdomain that interacts with the template strand (20), are highly conserved in enzymes related to DNA polymerase I.

Figure 2
DNA polymerase I amino acid sequence alignment. DNA polymerase I amino acid sequence alignment was generated with CLUSTAL W1.8. (34). GenBank accession nos as follows: Bastille ( ...

Intron sequence and structure

Figure Figure33 shows the putative secondary structures of the Bastille intron, which follows the group I consensus pairings P1 through P9, with dispensable pairing P2 missing (21). Based on the presence of extra nucleotides between P3 and P7, and conserved nucleotides in and adjacent to P7, the intron belongs to subgroup IA (22) within which most bacteriophage members comprise a distinct subgroup, IA2. Despite being inserted at a homologous site, the Bastille intron has significant differences in its secondary structures from the intron in SPO1 (and SP82). The Bastille intron lacks secondary structures P3.1 and P3.2, non-conserved structure elements that are inserted in the SPO1/SP82 introns between P3 and P4. The Bastille intron has only a single pairing element (P7.1) between P7 and P3, characteristic of the IA1 subfamily, rather than two elements (P7.1 and P7.2) that are typical of most phage introns (22). Finally, the Bastille intron has, typical for phage introns, a 7 bp P9 (terminating in a tetraloop) and a stem–loop P9.1, whereas the SPO1/SP82 introns have a single P9 with an elongated base-paired stem.

Figure 3
Secondary structures of Bastille (left) and SPO1 (right) intron. Exon sequences are in lower case and intron sequences in uppercase letters. Arrows indicate 5′ and 3′ splice sites (ss). Conserved structural elements P1 through P9 are ...

The Bastille intron encodes a nicking DNA endonuclease

Like most other phage introns, the Bastille intron has extra nucleotides inserted into the terminal loop of a conserved secondary structure element. In this case an open reading frame (ORF) of 188 codons (Supplementary Material, Fig. S1, residues 880–1443), which is preceded by a ribosome-binding site, is inserted into the loop of P8. This is the same location where the SPO1 and SP82 introns encode endonucleases I-HmuI and I-HmuII, respectively. In BLASTP searches (19) the Bastille intronic ORF was most similar to I-HmuI (E = 3e–24), I-HmuII (E = 4e–17) and several other phage-encoded proteins with the conserved H-N-H homing endonuclease motif. An alignment of the amino acid sequences is shown in Figure Figure44.

Figure 4
Amino acid sequence alignment of I-BasI with related phage H-N-H DNA endonucleases and C-termini of GIY-YIG endonucleases. Alignment was generated with CLUSTAL W1.8 (34) using amino acid sequences with GenBank accession nos as follows: I-BasI ( ...

A reverse position-specific BLAST (19) further indicated similarity to two conserved domains in the NCBI Conserved Domain Database (CDD) (23): the H-N-H motif (pfam01844) and, interestingly, to a domain described as intron-encoded nuclease repeat motif (IENR1: smart00497) possibly with a helix–turn–helix motif (HTH). In addition to H-N-H proteins, several GIY-YIG endonucleases have been identified as containing the conserved IENR1 domain. To further investigate the distribution of the conserved domain, protein database searches were employed with a motif search tool (MAST) (24) using an alignment comprising the IENR1 of phage H-N-H proteins shown in Figure Figure4.4. Among proteins containing the IENR1 motif, a weak similarity (E = 1.8) was identified to the C-terminal portion of I-BmoI, a group I intron-encoded homing endonuclease of the GIY-YIG family encoded by the thymidylate synthase intron in Bacillus mojavensis (25).

To address the question of whether the Bastille intron ORF encodes a functional endonuclease, the protein was expressed in E.coli for DNA endonuclease assays. Intron-encoded DNA endonucleases, in general, recognize and cleave the intronless version of their cognate genes. Thus, a DNA fragment, differentially end-labeled on either strand and containing part of the intron-minus Bastille DNA polymerase gene around the intron insertion site, was used as a substrate. Figure Figure55 shows that protein from cells expressing the intronic ORF has endonucleolytic activity, cleaving the intronless Bastille DNA polymerase gene. No activity was detected using protein derived from cells harboring the expression plasmid without insert. Based on this activity the intron-encoded ORF was designated I-BasI. Like I-HmuI and I-HmuII, I-BasI is a strand-specific endonuclease that introduces a nick in the template strand of the target DNA. No cleavage of the coding strand was detected under conditions in which the template strand was completely turned into product.

Figure 5
DNA endonuclease assay. Differentially end-labeled PCR products (either top or bottom strand, indicated by asterisk) were generated by amplification of the intron-minus and intron-plus Bastille, and the intron-minus SPO1 DNA polymerase gene as indicated. ...

Precise mapping of the cleavage sites on the Bastille and SPO1 substrates place the sites on the template strand 3 nt downstream of the intron insertion site (Fig. (Fig.6).6). Interestingly, I-BasI and I-HmuI have identical cleavage sites, with I-HmuI also nicking the coding strand 3 nt downstream of the intron insertion site [this places the cleavage site 1 nt further 3′ than reported previously in (12)]. The fact that both enzymes cleave at the same position suggests that they bind homologous stretches of their respective DNA polymerase genes.

Figure 6
I-BasI and I-HmuI cleavage site mapping. PCR products, end-labeled on the template strand, were generated from Bastille and SPO1 intronless DNA polymerase genes. DNA was incubated with (A) protein from cells expressing I-BasI (2), from cells harboring ...

Since I-HmuI also cleaves the intron-plus gene (12), a labeled DNA fragment containing the intron–exon II boundary was incubated with I-BasI (Fig. (Fig.5).5). However, unlike I-HmuI, I-BasI only cleaved the intronless gene. In a separate experiment (not shown) I-BasI was unable to cleave internally labeled PCR products that spanned the 5′ and 3′ splice sites, respectively. Surprisingly, while unable to nick the intron-containing Bastille sequence, I-BasI was able to cleave the intron-minus SPO1 gene despite considerable differences in the nucleotide sequence surrounding the cleavage site (Fig. (Fig.66C).

DISCUSSION

This work describes a self-splicing group I intron and intronic DNA endonuclease in the genome of B.thuringiensis phage Bastille. The intron is inserted in the DNA polymerase gene at the homologous position as the introns in SPO1, SP82 and Φe, all closely related phages infecting B.subtilis and containing the modified base hydroxymethyluracil (HMU) in place of thymine in their DNA (26). Bastille is morphologically distinct from the HMU phages (27) and its DNA is not extensively modified (our unpublished observation), suggesting that these Bacillus phages are unrelated. Like other group I introns (2,13), the insertion site is located in a highly conserved region of functional importance within the coding sequence (Fig. (Fig.22).

All four DNA polymerase introns have insertions in the loop of pairing element P8 that encode DNA endonucleases of the H-N-H family. The Bastille DNA polymerase and its intron-encoded endonuclease, I-BasI, are most similar to the SPO1 DNA polymerase and intron endonuclease I-HmuI, respectively, as indicated by being each other’s best match in the protein database. However, the structurally conserved portions of the introns differ substantially in sequence and secondary structure, arguing that the intron sequences do not share a recent common ancestor. Interestingly, the closest matches to the Bastille intron in a BLASTN search are the introns in Staphylococcus phage Twort (13,17).

A BLASTP search of the protein database using the I-BasI amino acid sequence revealed, in addition to I-HmuI, similarity to the intron-encoded endonuclease I-HmuII and several additional intronic and free-standing phage ORFs. The similarity among these proteins is most pronounced in the N-terminal part, whereas the C-termini have diverged considerably. The high conservation of the N-terminal region, which includes the H-N-H motif (which has been suggested to form the active site of these endonucleases), led to the proposal that these phage endonucleases have a two-domain structure (13,25) analogous to that of I-TevI, with N-terminal catalytic and C-terminal DNA-binding domains (7).

Database searches with the I-BasI sequence further indicated a similarity of the proposed C-terminal DNA-binding domain to a 53 amino acid consensus sequence described as IENR1 with a potential HTH (23). Surprisingly, more rigorous database searches identified a sequence similarity of the IENR1 motif to the C-terminal DNA-binding domain of B.mojavensis thyA intronic DNA endonuclease I-BmoI (25), which is highly similar to the structurally well-characterized phage endonuclease I-TevI (8,9). Sequence comparison of the C-termini of I-BmoI and I-TevI with the phage H-N-H endonucleases suggests that the IENR1 motif would make up a HTH subdomain with globular ββααβ fold, which is consistent with the secondary structure predication of the IENR1 consensus sequence (Fig. (Fig.4).4). In the crystal structure of the I-TevI C-terminus bound to DNA, the ββααβ fold represents a small globular DNA-binding module. Unlike traditional HTH domains, the HTH DNA-binding module of I-TevI contacts the major groove via phosphate backbone and hydrophobic interactions rather than base-specific contacts (8). The globular fold and the potential versatility in contacting DNA sequences in a site-specific and sequence-tolerant manner suggest that the IENR1 motif is well suited be fused to a catalytic cartridge like the H-N-H motif.

Similarity between proteins of the GIY-YIG and H-N-H families has been noted previously. The phage T4 protein MobD, a putative mobile element with an H-N-H motif at its N-terminus (28), can be aligned with another T4 protein, SegF, a member of the GIY-YIG family, over about 90 amino acids at their C-termini (29). The relatedness of H-N-H and GIY-YIG endonucleases in distinct sequence elements indicates the modular nature of these proteins. The shuffling of modules of protein structure, for example in the DNA-binding domain, could provide a mode for homing endonucleases to acquire new DNA substrate specificities.

While shuffling of DNA-binding modules is one potential pathway to generating new DNA recognition specificity, the phage intron-encoded endonucleases I-BasI, I-HmuI and I-HmuII provide an interesting example of how highly similar endonucleases have obtained distinct DNA substrate specificities without obvious domain shuffling. I-HmuI and I-HmuII have been shown previously to generate a nick in both intron-containing and intronless DNA, an activity and substrate specificity until then unseen for homing endonucleases, with each enzyme showing a preference for the DNA polymerase gene of the other phage (12). I-HmuII cleaves a site unrelated to the I-HmuI and I-BasI cleavage sites, 52 bp downstream of the intron insertion site, in a region of sequence heterogeneity with SPO1 DNA (12). In contrast to the unusual properties of these enzymes, I-BasI has the DNA substrate specificity of typical homing endonucleases. It cleaves the intronless Bastille DNA polymerase gene and, rather than cleaving the cognate intron-containing gene, it nicks the intronless SPO1 sequence despite nine differences in the 25-bp binding region (Fig. (Fig.4;4; M. Landthaler and D. A. Shub, unpublished data). Tolerance for cleaving homing sites with sequence variations has been described for several intron-encoded endonucleases: I-TevI (10), I-CreI (30,31), I-SceI (32) and I-PpoI (31,33).

The differences in DNA target specificities of I-BasI, I-HmuI and I-HmuII are likely a consequence of diverse evolutionary constraints. Goodrich-Blair and Shub (12) proposed that the unusual specificities of I-HmuI and I-HmuII might have arisen due to selective pressure to promote intron replacement rather than simple homing, due perhaps to a dearth of unoccupied homing sites. The existence of the closely related I-BasI, with properties expected for a typical homing endonuclease, may provide an interesting model to study how highly similar intronic endonucleases evolved to recognize and cleave distinct DNA substrates.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online.

[Supplementary Material]

ACKNOWLEDGEMENTS

We thank David Edgell and Rick Bonocora for critical reading of the manuscript. This work was supported by grant GM37746 from the National Institutes of Health.

REFERENCES

1. Lambowitz A.M. and Belfort,M. (1993) Introns as mobile genetic elements. Annu. Rev. Biochem., 62, 587–622. [PubMed]
2. Edgell D.R., Belfort,M. and Shub,D.A. (2000) Barriers to intron promiscuity in bacteria. J. Bacteriol., 182, 5281–5289. [PMC free article] [PubMed]
3. Belfort M. and Roberts,R.J. (1997) Homing endonucleases: keeping the house in order. Nucleic Acids Res., 25, 3379–3388. [PMC free article] [PubMed]
4. Chevalier B.S. and Stoddard,B.L. (2001) Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility. Nucleic Acids Res., 29, 3757–3774. [PMC free article] [PubMed]
5. Dalgaard J.Z., Klar,A.J., Moser,M.J., Holley,W.R., Chatterjee,A. and Mian,I.S. (1997) Statistical modeling and analysis of the LAGLIDADG family of site-specific endonucleases and identification of an intein that encodes a site-specific endonuclease of the HNH family. Nucleic Acids Res., 25, 4626–4638. [PMC free article] [PubMed]
6. Kowalski J.C., Belfort,M., Stapleton,M.A., Holpert,M., Dansereau,J.T., Pietrokovski,S., Baxter,S.M. and Derbyshire,V. (1999) Configuration of the catalytic GIY-YIG domain of intron endonuclease I-TevI: coincidence of computational and molecular findings. Nucleic Acids Res., 27, 2115–2125. [PMC free article] [PubMed]
7. Derbyshire V., Kowalski,J.C., Dansereau,J.T., Hauer,C.R. and Belfort,M. (1997) Two-domain structure of the td intron-encoded endonuclease I-TevI correlates with the two-domain configuration of the homing site. J. Mol. Biol., 265, 494–506. [PubMed]
8. Van Roey P., Waddling,C.A., Fox,K.M., Belfort,M. and Derbyshire,V. (2001) Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate. EMBO J., 20, 3631–3677. [PMC free article] [PubMed]
9. Van Roey P., Meehan,L., Kowalski,J.C., Belfort,M. and Derbyshire V. (2002) Catalytic domain structure and hypothesis for function of GIY-YIG intron endonuclease I-TevI. Nature Struct. Biol., 9, 806–811. [PubMed]
10. Bryk M., Quirk,S.M., Mueller,J.E., Loizos,N., Lawrence,C. and Belfort,M. (1993) The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target. EMBO J., 12, 4040–4041. [PMC free article] [PubMed]
11. Eddy S.R. and Gold,L. (1991) The phage T4 nrdB intron: a deletion mutant of a version found in the wild. Genes Dev., 5, 1032–1041. [PubMed]
12. Goodrich-Blair H. and Shub,D.A. (1996) Beyond homing: competition between intron endonucleases confers a selective advantage on flanking genetic markers. Cell, 84, 211–221. [PubMed]
13. Landthaler M., Begley,U., Lau,N.C. and Shub,D.A. (2002) Two self-splicing group I introns in the ribonucleotide reductase large subunit gene of Staphylococcus aureus phage Twort. Nucleic Acids Res., 30, 1935–1943. [PMC free article] [PubMed]
14. Perler F.B., Comb,D.G., Jack,W.E., Moran,L.S., Qiang,B., Kucera,R.B., Benner,J., Slatko,B.E., Nwankwo,P.O., Hepstead,S.K., Carlow,C.K.S. and Jannasch,H. (1992) Intervening sequences in an archaea DNA polymerase gene. Proc. Natl Acad. Sci. USA, 89, 5577–5581. [PMC free article] [PubMed]
15. Reinhold-Hurek B. and Shub,D.A. (1993) Experimental approaches for detecting self-splicing group I introns. In Zimmer,E.A., White,T.J., Cann,R.L. and Wilson,A.C. (eds), Methods Enzymology. Academic Press, San Diego, CA, Vol. 224, pp. 491–502. [PubMed]
16. Sambrook J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
17. Landthaler M. and Shub,D.A. (1999) Unexpected abundance of self-splicing introns in the genome of bacteriophage Twort: introns in multiple genes, a single gene with three introns and exon skipping by group I ribozymes. Proc. Natl Acad. Sci. USA, 96, 7005–7010. [PMC free article] [PubMed]
18. Bechhofer D.H., Hue,K.K. and Shub,D.A. (1994) An intron in the thymidylate synthase gene of Bacillus bacteriophage β22: evidence for independent evolution of a gene, its group I intron and the intron open reading frame. Proc. Natl Acad. Sci. USA, 91, 11669–11673. [PMC free article] [PubMed]
19. Altschul S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res., 25, 3389–3402. [PMC free article] [PubMed]
20. Patel P.H., Suzuki,M., Adman,E., Shinkai,A. and Loeb,L.A. (2001) Prokaryotic DNA polymerase I: evolution, structure and ‘base flipping’ mechanism for nucleotide selection. J. Mol. Biol., 308, 823–837. [PubMed]
21. Cech T.R., Damberger,S.H. and Gutell,R.R. (1994) Representation of the secondary and tertiary structure of group I introns. Nature Struct. Biol., 1, 273–280. [PubMed]
22. Michel F. and Westhof,E. (1990) Modeling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J. Mol. Biol., 216, 585–610. [PubMed]
23. Marchler-Bauer A., Anderson,J.B., DeWeese-Scott,C., Fedorova,N.D., Geer,L.Y., He,S., Hurwitz,D.I., Jackson,J.D., Jacobs,A.R., Lanczycki,C.J., Liebert,C.A., Liu,C., Madej,T., Marchler,G.H., Mazumder,R., Nikolskaya,A.N., Panchenko,A.R., Rao,B.S., Shoemaker,B.A., Simonyan,V., Song,J.S., Thiessen,P.A., Vasudevan,S., Wang,Y., Yamashita,R.A., Yin,J.J. and Bryant,S.H. (2003) CDD: a curated Entrez database of conserved domain alignments. Nucleic Acids Res., 31, 381–387.
24. Bailey T.L. and Gribskov,M. (1998) Combining evidence using p-values: application to sequence homology searches. Bioinformatics, 14, 48–54. [PubMed]
25. Edgell D.R. and Shub,D.A. (2001) Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites. Proc. Natl Acad. Sci. USA, 98, 7898–7903. [PMC free article] [PubMed]
26. Goodrich-Blair H. and Shub,D.A. (1994) The DNA polymerase genes of several HMU-bacteriophages have similar group I introns with highly divergent open reading frames. Nucleic Acids Res., 22, 3715–3721. [PMC free article] [PubMed]
27. Ackermann H.-W. and DuBow,M.S. (1987) Viruses of prokaryotes. Volume II: Natural Groups of Bacteriophages. CRC Press. Boca Raton, FL.
28. Kutter E., Gachechiladze,K., Poglazov,A., Marusich,E., Shneider,M., Aronsson,P., Napuli,A., Porter,D. and Mesyanzhinov,V. (1995) Evolution of T4-related phages. Virus Genes, 11, 285–297. [PubMed]
29. Mosig G., Colowick,N.E. and Pietz,B.C. (1998) Several new bacteriophage T4 genes, mapped by sequencing deletion endpoints between genes 56 (dCTPase) and dda (a DNA-dependent ATPase-helicase) modulate transcription. Gene, 223, 143–155. [PubMed]
30. Durrenberger F. and Rochaix,J.D. (1993) Characterization of the cleavage site and the recognition sequence of the I-CreI DNA endonuclease encoded by the chloroplast ribosomal intron of Chlamydomonas reinhardtii. Mol. Gen. Genet., 236, 409–414. [PubMed]
31. Argast G.M., Stephens,K.M., Emond,M.J. and Monnat,R.J.,Jr (1998) I-PpoI and I-CreI homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment. J. Mol. Biol., 280, 345–353. [PubMed]
32. Colleaux L., D’Auriol,L., Galibert,F. and Dujon,B. (1988) Recognition and cleavage site of the intron-encoded omega transposase. Proc. Natl Acad. Sci. USA, 85, 6022–6026. [PMC free article] [PubMed]
33. Wittmayer P.K., McKenzie,J.L. and Raines,R.T. (1998) Degenerate DNA recognition by I-PpoI endonuclease. Gene, 206, 11–21. [PubMed]
34. Thompson J.D., Higgins,D.G. and Gibson,T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22, 4673–4680. [PMC free article] [PubMed]
35. Jones D.T. (1999) Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol., 292, 195–202. [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...