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. 1992 Dec 11;20(23):6401–6407. doi: 10.1093/nar/20.23.6401

The I-CeuI endonuclease recognizes a sequence of 19 base pairs and preferentially cleaves the coding strand of the Chlamydomonas moewusii chloroplast large subunit rRNA gene.

P Marshall 1, C Lemieux 1
PMCID: PMC334533  PMID: 1475201

Abstract

The I-CeuI endonuclease is a member of the growing family of homing endonucleases that catalyse mobility of group I introns by making a double-strand break at the homing site of these introns in cognate intronless alleles during genetic crosses. In a previous study, we have shown that a short DNA fragment of 26 bp, encompassing the homing site of the fifth intron in the Chlamydomonas eugametos chloroplast large subunit rRNA gene (Ce LSU.5), was sufficient for I-CeuI recognition and cleavage. Here, we report the recognition sequence of the I-CeuI endonuclease, as determined by random mutagenesis of nucleotide positions adjacent to the I-CeuI cleavage site. Single-base substitutions that completely abolish endonuclease activity delimit a 15-bp sequence whereas those that reduce the cleavage rate define a 19-bp sequence that extends from position -7 to position +12 with respect to the Ce LSU.5 intron insertion site. As the other homing endonucleases that have been studied so far, the I-CeuI endonuclease recognizes a non-symmetric degenerate sequence. The top strand of the recognition sequence is preferred for I-CeuI cleavage and the bottom strand most likely determines the rate of double-strand breaks.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. 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]
  3. 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]
  4. Colleaux L., Michel-Wolwertz M. R., Matagne R. F., Dujon B. The apocytochrome b gene of Chlamydomonas smithii contains a mobile intron related to both Saccharomyces and Neurospora introns. Mol Gen Genet. 1990 Sep;223(2):288–296. doi: 10.1007/BF00265065. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Craik C. S., Largman C., Fletcher T., Roczniak S., Barr P. J., Fletterick R., Rutter W. J. Redesigning trypsin: alteration of substrate specificity. Science. 1985 Apr 19;228(4697):291–297. doi: 10.1126/science.3838593. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Dujon B. Sequence of the intron and flanking exons of the mitochondrial 21S rRNA gene of yeast strains having different alleles at the omega and rib-1 loci. Cell. 1980 May;20(1):185–197. doi: 10.1016/0092-8674(80)90246-9. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Gott J. M., Zeeh A., Bell-Pedersen D., Ehrenman K., Belfort M., Shub D. A. Genes within genes: independent expression of phage T4 intron open reading frames and the genes in which they reside. Genes Dev. 1988 Dec;2(12B):1791–1799. doi: 10.1101/gad.2.12b.1791. [DOI] [PubMed] [Google Scholar]
  13. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  14. Lemieux C., Lee R. W. Nonreciprocal recombination between alleles of the chloroplast 23S rRNA gene in interspecific Chlamydomonas crosses. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4166–4170. doi: 10.1073/pnas.84.12.4166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. Nunes-Düby S. E., Matsumoto L., Landy A. Half-att site substrates reveal the homology independence and minimal protein requirements for productive synapsis in lambda excisive recombination. Cell. 1989 Oct 6;59(1):197–206. doi: 10.1016/0092-8674(89)90881-7. [DOI] [PubMed] [Google Scholar]
  22. Perlman P. S., Butow R. A. Mobile introns and intron-encoded proteins. Science. 1989 Dec 1;246(4934):1106–1109. doi: 10.1126/science.2479980. [DOI] [PubMed] [Google Scholar]
  23. Quirk S. M., Bell-Pedersen D., Belfort M. Intron mobility in the T-even phages: high frequency inheritance of group I introns promoted by intron open reading frames. Cell. 1989 Feb 10;56(3):455–465. doi: 10.1016/0092-8674(89)90248-1. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Szostak J. W., Orr-Weaver T. L., Rothstein R. J., Stahl F. W. The double-strand-break repair model for recombination. Cell. 1983 May;33(1):25–35. doi: 10.1016/0092-8674(83)90331-8. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Vollrath D., Davis R. W., Connelly C., Hieter P. Physical mapping of large DNA by chromosome fragmentation. Proc Natl Acad Sci U S A. 1988 Aug;85(16):6027–6031. doi: 10.1073/pnas.85.16.6027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. West D. K., Changchien L. M., Maley G. F., Maley F. Evidence that the intron open reading frame of the phage T4 td gene encodes a specific endonuclease. J Biol Chem. 1989 Jun 25;264(18):10343–10346. [PubMed] [Google Scholar]

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