• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of embojLink to Publisher's site
EMBO J. Jul 1993; 12(7): 2939–2947.
PMCID: PMC413549

Asymmetrical recognition and activity of the I-SceI endonuclease on its site and on intron-exon junctions.

Abstract

Group I intron-encoded endonucleases represent a new class of double strand cutting endonucleases whose function is to initiate the homing of introns by generating double strand breaks in site-specific sequences. We have studied the mechanism of interaction of the I-SceI endonuclease with different DNA substrates derived from its natural site in the intron-less gene or from intron-exon junctions in the gene with an intron. We show that the enzyme recognizes its asymmetrical site with high affinity binding to the sequence corresponding to the downstream exon followed by binding to the upstream exon and catalysis of phosphodiester bond hydrolysis. Asymmetrical nicking activity is observed as an intermediate of the cleavage reaction. In the intron-containing gene, the enzyme recognizes the downstream intron-exon junction without any cleavage activity. This binding raises the possibility of a specific function of homing endonucleases in either gene expression or intron homing steps subsequent to DNA cleavage.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Belfort M. Self-splicing introns in prokaryotes: migrant fossils? Cell. 1991 Jan 11;64(1):9–11. [PubMed]
  • Bell-Pedersen D, Quirk S, Clyman J, Belfort M. Intron mobility in phage T4 is dependent upon a distinctive class of endonucleases and independent of DNA sequences encoding the intron core: mechanistic and evolutionary implications. Nucleic Acids Res. 1990 Jul 11;18(13):3763–3770. [PMC free article] [PubMed]
  • Bennett SP, Halford SE. Recognition of DNA by type II restriction enzymes. Curr Top Cell Regul. 1989;30:57–104. [PubMed]
  • Bremer MC, Gimble FS, Thorner J, Smith CL. VDE endonuclease cleaves Saccharomyces cerevisiae genomic DNA at a single site: physical mapping of the VMA1 gene. Nucleic Acids Res. 1992 Oct 25;20(20):5484–5484. [PMC free article] [PubMed]
  • Buckle M, Geiselmann J, Kolb A, Buc H. Protein-DNA cross-linking at the lac promoter. Nucleic Acids Res. 1991 Feb 25;19(4):833–840. [PMC free article] [PubMed]
  • Busch SJ, Sassone-Corsi P. Dimers, leucine zippers and DNA-binding domains. Trends Genet. 1990 Feb;6(2):36–40. [PubMed]
  • Cech TR. Self-splicing of group I introns. Annu Rev Biochem. 1990;59:543–568. [PubMed]
  • Chu FK, Maley G, Pedersen-Lane J, Wang AM, Maley F. Characterization of the restriction site of a prokaryotic intron-encoded endonuclease. Proc Natl Acad Sci U S A. 1990 May;87(9):3574–3578. [PMC free article] [PubMed]
  • Colleaux L, d'Auriol L, Betermier M, Cottarel G, Jacquier A, Galibert F, Dujon B. Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease. Cell. 1986 Feb 28;44(4):521–533. [PubMed]
  • Colleaux L, D'Auriol L, Galibert F, Dujon B. Recognition and cleavage site of the intron-encoded omega transposase. Proc Natl Acad Sci U S A. 1988 Aug;85(16):6022–6026. [PMC free article] [PubMed]
  • Davis EO, Jenner PJ, Brooks PC, Colston MJ, Sedgwick SG. Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence. Cell. 1992 Oct 16;71(2):201–210. [PubMed]
  • Delahodde A, Goguel V, Becam AM, Creusot F, Perea J, Banroques J, Jacq C. Site-specific DNA endonuclease and RNA maturase activities of two homologous intron-encoded proteins from yeast mitochondria. Cell. 1989 Feb 10;56(3):431–441. [PubMed]
  • Dujon B. Group I introns as mobile genetic elements: facts and mechanistic speculations--a review. Gene. 1989 Oct 15;82(1):91–114. [PubMed]
  • Dujon B, Belfort M, Butow RA, Jacq C, Lemieux C, Perlman PS, Vogt VM. Mobile introns: definition of terms and recommended nomenclature. Gene. 1989 Oct 15;82(1):115–118. [PubMed]
  • Dürrenberger F, Rochaix JD. Chloroplast ribosomal intron of Chlamydomonas reinhardtii: in vitro self-splicing, DNA endonuclease activity and in vivo mobility. EMBO J. 1991 Nov;10(11):3495–3501. [PMC free article] [PubMed]
  • Eddy SR, Gold L. The phage T4 nrdB intron: a deletion mutant of a version found in the wild. Genes Dev. 1991 Jun;5(6):1032–1041. [PubMed]
  • Gauthier A, Turmel M, Lemieux C. A group I intron in the chloroplast large subunit rRNA gene of Chlamydomonas eugametos encodes a double-strand endonuclease that cleaves the homing site of this intron. Curr Genet. 1991 Jan;19(1):43–47. [PubMed]
  • Gimble FS, Thorner J. Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae. Nature. 1992 May 28;357(6376):301–306. [PubMed]
  • Goguel V, Delahodde A, Jacq C. Connections between RNA splicing and DNA intron mobility in yeast mitochondria: RNA maturase and DNA endonuclease switching experiments. Mol Cell Biol. 1992 Feb;12(2):696–705. [PMC free article] [PubMed]
  • Harrison SC, Aggarwal AK. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem. 1990;59:933–969. [PubMed]
  • Heitman J. How the EcoRI endonuclease recognizes and cleaves DNA. Bioessays. 1992 Jul;14(7):445–454. [PubMed]
  • Hirata R, Ohsumk Y, Nakano A, Kawasaki H, Suzuki K, Anraku Y. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem. 1990 Apr 25;265(12):6726–6733. [PubMed]
  • Hockensmith JW, Kubasek WL, Vorachek WR, Evertsz EM, von Hippel PH. Laser cross-linking of protein-nucleic acid complexes. Methods Enzymol. 1991;208:211–236. [PubMed]
  • Hodges RA, Perler FB, Noren CJ, Jack WE. Protein splicing removes intervening sequences in an archaea DNA polymerase. Nucleic Acids Res. 1992 Dec 11;20(23):6153–6157. [PMC free article] [PubMed]
  • Kawasaki K, Takahashi M, Natori M, Shibata T. DNA sequence recognition by a eukaryotic sequence-specific endonuclease, Endo.SceI, from Saccharomyces cerevisiae. J Biol Chem. 1991 Mar 15;266(8):5342–5347. [PubMed]
  • Kostriken R, Strathern JN, Klar AJ, Hicks JB, Heffron F. A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae. Cell. 1983 Nov;35(1):167–174. [PubMed]
  • Lambowitz AM. Infectious introns. Cell. 1989 Feb 10;56(3):323–326. [PubMed]
  • Lesser DR, Kurpiewski MR, Jen-Jacobson L. The energetic basis of specificity in the Eco RI endonuclease--DNA interaction. Science. 1990 Nov 9;250(4982):776–786. [PubMed]
  • Ma DP, King YT, Kim Y, Luckett WS., Jr The group I intron of apocytochrome b gene from Chlamydomonas smithii encodes a site-specific endonuclease. Plant Mol Biol. 1992 Mar;18(5):1001–1004. [PubMed]
  • Marshall P, Lemieux C. Cleavage pattern of the homing endonuclease encoded by the fifth intron in the chloroplast large subunit rRNA-encoding gene of Chlamydomonas eugametos. Gene. 1991 Aug 15;104(2):241–245. [PubMed]
  • Monteilhet C, Perrin A, Thierry A, Colleaux L, Dujon B. Purification and characterization of the in vitro activity of I-Sce I, a novel and highly specific endonuclease encoded by a group I intron. Nucleic Acids Res. 1990 Mar 25;18(6):1407–1413. [PMC free article] [PubMed]
  • Moran JV, Wernette CM, Mecklenburg KL, Butow RA, Perlman PS. Intron 5 alpha of the COXI gene of yeast mitochondrial DNA is a mobile group I intron. Nucleic Acids Res. 1992 Aug 11;20(15):4069–4076. [PMC free article] [PubMed]
  • Morishima N, Nakagawa K, Yamamoto E, Shibata T. A subunit of yeast site-specific endonuclease SceI is a mitochondrial version of the 70-kDa heat shock protein. J Biol Chem. 1990 Sep 5;265(25):15189–15197. [PubMed]
  • Muscarella DE, Vogt VM. A mobile group I intron in the nuclear rDNA of Physarum polycephalum. Cell. 1989 Feb 10;56(3):443–454. [PubMed]
  • Muscarella DE, Ellison EL, Ruoff BM, Vogt VM. Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum. Mol Cell Biol. 1990 Jul;10(7):3386–3396. [PMC free article] [PubMed]
  • Nakagawa K, Morishima N, Shibata T. A maturase-like subunit of the sequence-specific endonuclease endo.SceI from yeast mitochondria. J Biol Chem. 1991 Jan 25;266(3):1977–1984. [PubMed]
  • Nakagawa K, Morishima N, Shibata T. An endonuclease with multiple cutting sites, Endo.SceI, initiates genetic recombination at its cutting site in yeast mitochondria. EMBO J. 1992 Jul;11(7):2707–2715. [PMC free article] [PubMed]
  • Nickoloff JA, Singer JD, Heffron F. In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis. Mol Cell Biol. 1990 Mar;10(3):1174–1179. [PMC free article] [PubMed]
  • Partono S, Lewin AS. The rate and specificity of a group I ribozyme are inversely affected by choice of monovalent salt. Nucleic Acids Res. 1991 Feb 11;19(3):605–609. [PMC free article] [PubMed]
  • Perler FB, Comb DG, Jack WE, Moran LS, Qiang B, Kucera RB, Benner J, Slatko BE, Nwankwo DO, Hempstead SK, et al. Intervening sequences in an Archaea DNA polymerase gene. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5577–5581. [PMC free article] [PubMed]
  • Sargueil B, Hatat D, Delahodde A, Jacq C. In vivo and in vitro analyses of an intron-encoded DNA endonuclease from yeast mitochondria. Recognition site by site-directed mutagenesis. Nucleic Acids Res. 1990 Oct 11;18(19):5659–5665. [PMC free article] [PubMed]
  • Sargueil B, Delahodde A, Hatat D, Tian GL, Lazowska J, Jacq C. A new specific DNA endonuclease activity in yeast mitochondria. Mol Gen Genet. 1991 Feb;225(2):340–341. [PubMed]
  • Scazzocchio C. Group I introns: do they only go home? Trends Genet. 1989 Jun;5(6):168–172. [PubMed]
  • Séraphin B, Faye G, Hatat D, Jacq C. The yeast mitochondrial intron aI5 alpha: associated endonuclease activity and in vivo mobility. Gene. 1992 Apr 1;113(1):1–8. [PubMed]
  • Shibata T, Watabe H, Kaneko T, Iino T, Ando T. On the nucleotide sequence recognized by a eukaryotic site-specific endonuclease, Endo.SceI from yeast. J Biol Chem. 1984 Aug 25;259(16):10499–10506. [PubMed]
  • Shih CK, Wagner R, Feinstein S, Kanik-Ennulat C, Neff N. A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth. Mol Cell Biol. 1988 Aug;8(8):3094–3103. [PMC free article] [PubMed]
  • Shub DA, Goodrich-Blair H. Protein introns: a new home for endonucleases. Cell. 1992 Oct 16;71(2):183–186. [PubMed]
  • Takeda Y, Ohlendorf DH, Anderson WF, Matthews BW. DNA-binding proteins. Science. 1983 Sep 9;221(4615):1020–1026. [PubMed]
  • Thielking V, Alves J, Fliess A, Maass G, Pingoud A. Accuracy of the EcoRI restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences. Biochemistry. 1990 May 15;29(19):4682–4691. [PubMed]
  • Thierry A, Dujon B. Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes. Nucleic Acids Res. 1992 Nov 11;20(21):5625–5631. [PMC free article] [PubMed]
  • Thierry A, Perrin A, Boyer J, Fairhead C, Dujon B, Frey B, Schmitz G. Cleavage of yeast and bacteriophage T7 genomes at a single site using the rare cutter endonuclease I-Sce I. Nucleic Acids Res. 1991 Jan 11;19(1):189–190. [PMC free article] [PubMed]
  • Thompson AJ, Yuan X, Kudlicki W, Herrin DL. Cleavage and recognition pattern of a double-strand-specific endonuclease (I-creI) encoded by the chloroplast 23S rRNA intron of Chlamydomonas reinhardtii. Gene. 1992 Oct 1;119(2):247–251. [PubMed]
  • Wenzlau JM, Saldanha RJ, Butow RA, Perlman PS. A latent intron-encoded maturase is also an endonuclease needed for intron mobility. Cell. 1989 Feb 10;56(3):421–430. [PubMed]
  • Wernette CM, Saldahna R, Perlman PS, Butow RA. Purification of a site-specific endonuclease, I-Sce II, encoded by intron 4 alpha of the mitochondrial coxI gene of Saccharomyces cerevisiae. J Biol Chem. 1990 Nov 5;265(31):18976–18982. [PubMed]
  • Wernette C, Saldanha R, Smith D, Ming D, Perlman PS, Butow RA. Complex recognition site for the group I intron-encoded endonuclease I-SceII. Mol Cell Biol. 1992 Feb;12(2):716–723. [PMC free article] [PubMed]
  • Wilson GG, Murray NE. Restriction and modification systems. Annu Rev Genet. 1991;25:585–627. [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • Gene
    Gene
    Gene links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • MedGen
    MedGen
    Related information in MedGen
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...