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. 1993 Jul;12(7):2939–2947. doi: 10.1002/j.1460-2075.1993.tb05956.x

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

A Perrin 1, M Buckle 1, B Dujon 1
PMCID: PMC413549  PMID: 8335007

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.

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Selected References

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  1. Belfort M. Self-splicing introns in prokaryotes: migrant fossils? Cell. 1991 Jan 11;64(1):9–11. doi: 10.1016/0092-8674(91)90201-9. [DOI] [PubMed] [Google Scholar]
  2. 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. doi: 10.1093/nar/18.13.3763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett S. P., Halford S. E. Recognition of DNA by type II restriction enzymes. Curr Top Cell Regul. 1989;30:57–104. doi: 10.1016/b978-0-12-152830-0.50005-0. [DOI] [PubMed] [Google Scholar]
  4. Bremer M. C., Gimble F. S., Thorner J., Smith C. L. 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. doi: 10.1093/nar/20.20.5484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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. doi: 10.1093/nar/19.4.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Busch S. J., Sassone-Corsi P. Dimers, leucine zippers and DNA-binding domains. Trends Genet. 1990 Feb;6(2):36–40. doi: 10.1016/0168-9525(90)90071-d. [DOI] [PubMed] [Google Scholar]
  7. Cech T. R. Self-splicing of group I introns. Annu Rev Biochem. 1990;59:543–568. doi: 10.1146/annurev.bi.59.070190.002551. [DOI] [PubMed] [Google Scholar]
  8. Chu F. K., Maley G., Pedersen-Lane J., Wang A. M., 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. doi: 10.1073/pnas.87.9.3574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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. doi: 10.1073/pnas.85.16.6022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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. doi: 10.1016/0092-8674(86)90262-x. [DOI] [PubMed] [Google Scholar]
  11. Davis E. O., Jenner P. J., Brooks P. C., Colston M. J., Sedgwick S. G. 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. doi: 10.1016/0092-8674(92)90349-h. [DOI] [PubMed] [Google Scholar]
  12. Delahodde A., Goguel V., Becam A. M., 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. doi: 10.1016/0092-8674(89)90246-8. [DOI] [PubMed] [Google Scholar]
  13. Dujon B., Belfort M., Butow R. A., Jacq C., Lemieux C., Perlman P. S., Vogt V. M. Mobile introns: definition of terms and recommended nomenclature. Gene. 1989 Oct 15;82(1):115–118. doi: 10.1016/0378-1119(89)90035-8. [DOI] [PubMed] [Google Scholar]
  14. Dujon B. Group I introns as mobile genetic elements: facts and mechanistic speculations--a review. Gene. 1989 Oct 15;82(1):91–114. doi: 10.1016/0378-1119(89)90034-6. [DOI] [PubMed] [Google Scholar]
  15. Dürrenberger F., Rochaix J. D. 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. doi: 10.1002/j.1460-2075.1991.tb04913.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Eddy S. R., 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. doi: 10.1101/gad.5.6.1032. [DOI] [PubMed] [Google Scholar]
  17. 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. doi: 10.1007/BF00362086. [DOI] [PubMed] [Google Scholar]
  18. Gimble F. S., Thorner J. Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae. Nature. 1992 May 28;357(6376):301–306. doi: 10.1038/357301a0. [DOI] [PubMed] [Google Scholar]
  19. 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. doi: 10.1128/mcb.12.2.696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harrison S. C., Aggarwal A. K. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem. 1990;59:933–969. doi: 10.1146/annurev.bi.59.070190.004441. [DOI] [PubMed] [Google Scholar]
  21. Heitman J. How the EcoRI endonuclease recognizes and cleaves DNA. Bioessays. 1992 Jul;14(7):445–454. doi: 10.1002/bies.950140704. [DOI] [PubMed] [Google Scholar]
  22. 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] [Google Scholar]
  23. Hockensmith J. W., Kubasek W. L., Vorachek W. R., Evertsz E. M., von Hippel P. H. Laser cross-linking of protein-nucleic acid complexes. Methods Enzymol. 1991;208:211–236. doi: 10.1016/0076-6879(91)08015-a. [DOI] [PubMed] [Google Scholar]
  24. Hodges R. A., Perler F. B., Noren C. J., Jack W. E. Protein splicing removes intervening sequences in an archaea DNA polymerase. Nucleic Acids Res. 1992 Dec 11;20(23):6153–6157. doi: 10.1093/nar/20.23.6153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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] [Google Scholar]
  26. Kostriken R., Strathern J. N., Klar A. J., Hicks J. B., Heffron F. A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae. Cell. 1983 Nov;35(1):167–174. doi: 10.1016/0092-8674(83)90219-2. [DOI] [PubMed] [Google Scholar]
  27. Lambowitz A. M. Infectious introns. Cell. 1989 Feb 10;56(3):323–326. doi: 10.1016/0092-8674(89)90232-8. [DOI] [PubMed] [Google Scholar]
  28. Lesser D. R., Kurpiewski M. R., Jen-Jacobson L. The energetic basis of specificity in the Eco RI endonuclease--DNA interaction. Science. 1990 Nov 9;250(4982):776–786. doi: 10.1126/science.2237428. [DOI] [PubMed] [Google Scholar]
  29. Ma D. P., King Y. T., Kim Y., Luckett W. S., 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. doi: 10.1007/BF00019218. [DOI] [PubMed] [Google Scholar]
  30. 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. doi: 10.1016/0378-1119(91)90256-b. [DOI] [PubMed] [Google Scholar]
  31. 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. doi: 10.1093/nar/18.6.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moran J. V., Wernette C. M., Mecklenburg K. L., Butow R. A., Perlman P. S. 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. doi: 10.1093/nar/20.15.4069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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] [Google Scholar]
  34. Muscarella D. E., Ellison E. L., Ruoff B. M., Vogt V. M. 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. doi: 10.1128/mcb.10.7.3386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Muscarella D. E., Vogt V. M. A mobile group I intron in the nuclear rDNA of Physarum polycephalum. Cell. 1989 Feb 10;56(3):443–454. doi: 10.1016/0092-8674(89)90247-x. [DOI] [PubMed] [Google Scholar]
  36. 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] [Google Scholar]
  37. 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. doi: 10.1002/j.1460-2075.1992.tb05336.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nickoloff J. A., Singer J. D., 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. doi: 10.1128/mcb.10.3.1174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Partono S., Lewin A. S. 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. doi: 10.1093/nar/19.3.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Perler F. B., Comb D. G., Jack W. E., Moran L. S., Qiang B., Kucera R. B., Benner J., Slatko B. E., Nwankwo D. O., Hempstead S. K. Intervening sequences in an Archaea DNA polymerase gene. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5577–5581. doi: 10.1073/pnas.89.12.5577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sargueil B., Delahodde A., Hatat D., Tian G. L., Lazowska J., Jacq C. A new specific DNA endonuclease activity in yeast mitochondria. Mol Gen Genet. 1991 Feb;225(2):340–341. doi: 10.1007/BF00269867. [DOI] [PubMed] [Google Scholar]
  42. 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. doi: 10.1093/nar/18.19.5659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Scazzocchio C. Group I introns: do they only go home? Trends Genet. 1989 Jun;5(6):168–172. doi: 10.1016/0168-9525(89)90068-1. [DOI] [PubMed] [Google Scholar]
  44. 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] [Google Scholar]
  45. Shih C. K., 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. doi: 10.1128/mcb.8.8.3094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shub D. A., Goodrich-Blair H. Protein introns: a new home for endonucleases. Cell. 1992 Oct 16;71(2):183–186. doi: 10.1016/0092-8674(92)90345-d. [DOI] [PubMed] [Google Scholar]
  47. 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. doi: 10.1016/0378-1119(92)90663-a. [DOI] [PubMed] [Google Scholar]
  48. Takeda Y., Ohlendorf D. H., Anderson W. F., Matthews B. W. DNA-binding proteins. Science. 1983 Sep 9;221(4615):1020–1026. doi: 10.1126/science.6308768. [DOI] [PubMed] [Google Scholar]
  49. 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. doi: 10.1021/bi00471a024. [DOI] [PubMed] [Google Scholar]
  50. 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. doi: 10.1093/nar/20.21.5625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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. doi: 10.1093/nar/19.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Thompson A. J., Yuan X., Kudlicki W., Herrin D. L. 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. doi: 10.1016/0378-1119(92)90278-w. [DOI] [PubMed] [Google Scholar]
  53. Wenzlau J. M., Saldanha R. J., Butow R. A., Perlman P. S. A latent intron-encoded maturase is also an endonuclease needed for intron mobility. Cell. 1989 Feb 10;56(3):421–430. doi: 10.1016/0092-8674(89)90245-6. [DOI] [PubMed] [Google Scholar]
  54. Wernette C. M., Saldahna R., Perlman P. S., Butow R. A. 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] [Google Scholar]
  55. Wernette C., Saldanha R., Smith D., Ming D., Perlman P. S., Butow R. A. Complex recognition site for the group I intron-encoded endonuclease I-SceII. Mol Cell Biol. 1992 Feb;12(2):716–723. doi: 10.1128/mcb.12.2.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wilson G. G., Murray N. E. Restriction and modification systems. Annu Rev Genet. 1991;25:585–627. doi: 10.1146/annurev.ge.25.120191.003101. [DOI] [PubMed] [Google Scholar]

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