Nucleic Acids Research, 2003, Vol. 31, No. 12 3071±3077 DOI: 10.1093/nar/gkg433 The nicking homing endonuclease I-BasI is encoded by a group I intron in the DNA polymerase gene of the Bacillus thuringiensis phage Bastille Markus Landthaler and David A. Shub* University at Albany, SUNY, Department of Biological Sciences and Center for Molecular Genetics, Albany, NY, USA Downloaded from https://academic.oup.com/nar/article/31/12/3071/1395342 by guest on 28 September 2021 Received March 10, 2003; Revised and Accepted May 2, 2003 ABSTRACT occupies in the intron containing homolog, by a duplicative and unidirectional transfer. Here we describe the discovery of a group I intron in Homing endonucleases belong to four families based on the the DNA polymerase gene of Bacillus thuringiensis presence of well-conserved sequence motifs that are denoted phage Bastille. Although the intron insertion site is LAGLIDADG, His-Cys box, GIY-YIG and H-N-H, respect- identical to that of the Bacillus subtilis phages ively (3). Most homing endonucleases bind DNA in a SPO1 and SP82 introns, the Bastille intron differs sequence-speci®c fashion, recognizing large stretches of the from them substantially in primary and secondary intron-minus version of the gene in which they are inserted. In structure. Like the SPO1 and SP82 introns, the general, the recognition site comprises sequences of both Bastille intron encodes a nicking DNA endo- exons and the endonucleases generate a double-strand break nuclease of the H-N-H family, I-BasI, with a cleavage close to the intron insertion site (4). Two of the families, the GIY-YIG and H-N-H homing site identical to that of the SPO1-encoded enzyme endonucleases, have been found in group I introns, and also as I-HmuI. Unlike I-HmuI, which nicks both intron- freestanding open-reading frames, in bacteria and their phages minus and intron-plus DNA, I-BasI cleaves only (2,5,6). The coliphage T4 td intron-encoded endonuclease intron-minus alleles, which is a characteristic of I-TevI is the best-studied member of the GIY-YIG family. It is typical homing endonucleases. Interestingly, the a bipartite enzyme with distinct catalytic and DNA-binding C-terminal portions of these H-N-H phage endo- domains (7±9) that recognizes a 37-bp region in a sequence nucleases contain a conserved sequence motif, the tolerant manner (10). Members of the H-N-H family are less intron-encoded endonuclease repeat motif (IENR1) well understood, as they possess biochemical activities usually that also has been found in endonucleases of the not associated with other homing endonucleases. For instance, I-TevIII of phage RB3 makes a double-strand break similar to GIY-YIG family, and which likely comprises a small other endonucleases but is the only homing endonuclease that DNA-binding module with a globular bbaab fold, generates a 5¢ overhang instead of the typical 3¢ extension suggestive of module shuf¯ing between different (11). The H-N-H endonucleases I-HmuI and I-HmuII, homing endonuclease families. 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 INTRODUCTION intron-plus as well as intron-minus alleles, and cut only one strand of their DNA substrate (12). Another example of a Group I introns are found in protein- and RNA-coding genes nicking H-N-H endonuclease, I-TwoI, is encoded by a group I of a diverse set of organisms. In eukaryotes, group I introns intron in the ribonucleotide reductase gene of staphylococcal exist in nuclear rRNA genes of fungi and ciliates and in phage Twort (13). organellar genes of plants, fungi and ¯agellates (1). They In this work we describe a group I intron in the DNA have also been found in cyanobacteria, proteobacteria, polymerase gene of Bacillus thuringiensis phage Bastille. Like Gram-positive bacteria and bacteriophages (2). the SPO1 and SP82 introns, the Bastille intron encodes a A large number of group I introns encode site-speci®c DNA nicking DNA endonuclease and its cleavage site is identical to endonucleases (1). These intron-encoded endonucleases that of the SPO1-encoded enzyme I-HmuI. However, in (homing endonucleases) function primarily in a biological contrast to I-HmuI, I-BasI has DNA substrate speci®city for process whereby a group I intron is inserted into an intron-minus alleles, which is characteristic of a typical intronless version of a gene, at the same position it already homing endonuclease. Sequence comparisons of the *To whom correspondence should be addressed. Tel: +1 518 442 4324; Fax: +1 518 442 4767; Email: [email protected] Present address: Markus Landthaler, Rockefeller University, New York, NY, USA Nucleic Acids Research, Vol. 31 No. 12 ã Oxford University Press 2003; all rights reserved 3072 Nucleic Acids Research, 2003, Vol. 31, No. 12 C-terminal portion of phage H-N-H endonucleases against the were separated on a 5% denaturing polyacrylamide gel and protein database indicate the presence of a conserved sequence visualized by autoradiography. motif that has also been found in endonucleases of the GIY- YIG family, likely forming a small DNA-binding module with Isolation of Bastille RNA a globular bbaab fold. Bacillus thuringiensis cells were grown at 37°CtoanOD540 of 0.4 (~5 3 107 cells/ml) and infected with ~5 phages/cell. Cells (25 ml) were harvested by centrifugation at 5000 g for 10 min MATERIALS AND METHODS at 4°C and washed twice with 10 mM Tris±HCl (pH 7.5 at 4°C), 100 mg/ml chloramphenicol. Cells were resuspended in Bacterial and bacteriophage strains 100 ml 10 mM Tris±HCl (pH 8.0), 1 mM EDTA, 50 mg/ml Phage Bastille (HER211) and its host B.thuringiensis lysozyme (Sigma) and incubated for 5 min at room tempera- (HER1211) were obtained from the Felix d'Herelle ture. RNA was isolated with the RNeasy Kit (QIAGEN) as Downloaded from https://academic.oup.com/nar/article/31/12/3071/1395342 by guest on 28 September 2021 Reference Center. Escherichia coli XL-1 Blue (Stratagene) explained in the manufacture's protocol for isolation of total was used as the recipient strain for high-frequency plasmid RNA from bacteria. In vitro labeling of RNA with [a-32P]GTP electroporation. Escherichia coli BL21(DE3) pLysE was used was according to Reinhold-Hurek and Shub (15). as the bacterial host for protein expression. Isolation of Bastille phage DNA Plasmids Bacillus thuringiensis cells were grown to an OD of ~0.4, The plasmid (pBET) for over-expression of I-BasI was 540 infected at a multiplicity of ~0.1 phage/cell, and incubation generated by PCR ampli®cation of Bastille DNA using was continued until lysis was complete. Phages were primers BIorf-U2 and BIorf-D. The PCR product was digested precipitated in 10% PEG 8000 and phage particles were with NdeI and ligated into the NdeI site of pAii17 (14). further puri®ed by centrifugation through a CsCl step gradient Expression and preparation of I-BasI protein extracts according to the bacteriophage l puri®cation protocol in Sambrook et al. (16). After removal of CsCl by dialysis Cells were grown in LB supplemented with ampicillin against 50 mM Tris±HCl (pH 8.0) and 1 mM EDTA, the phage (50 mg/ml) at 37°CtoA600 = 0.6 and expression was induced DNA was extracted with phenol. by addition of IPTG to a ®nal 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 Bastille DNA southern hybridization resuspended in ice-cold 50 mM Tris±HCl (pH 7.2), 1 mM Restriction enzyme digests of Bastille DNA were separated on EDTA, 1 mM PMSF, 2 mg/ml leupeptin, 200 mM KCl at a a 1% agarose gel and vacuum blotted onto a positively charged concentration of 6 ml/g cells. The resuspended cells were nylon membrane (Hybond-N+, Amersham) by alkaline trans- sonicated to complete lysis and centrifuged at 12 000 g for 1 h. fer. The Bastille DNA blot was probed with radiolabeled The protein was present in the pellet fraction. The pellet was phage Twort orf142 intron DNA (17). The probe was washed with chilled deionized H2O, resuspended in 1 ml/g generated by random primed labeling of a 992-bp PCR (cells) 6 M guanidine hydrochloride, renatured by dialyzing product, derived from ampli®cation of Twort DNA using twice against 1003 vol of 50 mM potassium phosphate primers 4 and 5 from Landthaler and Shub (17). Hybridization (pH 7.2), 100 mM NaCl, 1 mM DTT and stored in 10% was carried out in 63 SSC, 53 Denhardt's solution, 0.1% glycerol at ±80°C. SDS, 200 mg/ml herring sperm DNA. Four washes at 50°Cin Endonuclease assay with extracts from cells expressing 23 SSC, 0.1% SDS were followed by a single wash in I-BasI 0.2 3 SSC, 0.1% SDS at 50°C. PCR-generated DNA fragments, radio-labeled at their 5¢ termini, were used as substrates. The intron-minus Bastille Mapping of I-BasI cleavage sites DNA substrate was generated by reverse transcription of Intron-minus Bastille and SPO1 DNA substrates were gener- Bastille RNA isolated 10 min post-infection with primer ated as described above with 32P-labeled bottom strand S2373 and subsequent PCR ampli®cation with primers S2325 primers. Labeled PCR products were incubated with 1/10 vol and S2373. The intron-plus Bastille DNA substrate was PCR- of I-BasI protein extract in 50 mM Tris±HCl (pH 7.9), 10 mM generated by ampli®cation of Bastille DNA with primers MgCl2, 100 mM NaCl, 1 mM DTT, phenol extracted and S2327 and S2373.