Purification and Characterization of the DNA Cleavage and Recognition Site of I-ScaI Mitochondrial Group I Intron Encoded Endonuclease Produced in Escherichia Coli

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2000 Feb 10

PMID 10666469

Citations 7

Authors

C Monteilhet

D Dziadkowiec

T Szczepanek

J Lazowska

Affiliations

Soon will be listed here.

Abstract

The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs. Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain. It acts as a specific homing endonuclease I- Sca I promoting intron mobility and as a maturase promoting intron splicing. Here we describe the synthesis of a universal code equivalent to the mitochondrial sequence coding for this protein and the in vitro characterization of I- Sca I endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli. We have also determined the cleavage pattern as well as the recognition site of p28bi2. It was found that p28bi2 generates a double-strand cleavage downstream from the intron insertion site with 4 nt long 3'-overhangs. Mutational analysis of the DNA target site shows that p28bi2 recognizes a 16-19 bp sequence from positions -11 to +8 with respect to the intron insertion site.

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References

Jacq C, Banroques J, Becam A, SLONIMSKI P, Guiso N, Danchin A . Antibodies against a fused 'lacZ-yeast mitochondrial intron' gene product allow identification of the mRNA maturase encoded by the fourth intron of the yeast cob-box gene. EMBO J. 1984; 3(7):1567-72. PMC: 557560. DOI: 10.1002/j.1460-2075.1984.tb02012.x. View

Wang J, Kim H, Yuan X, Herrin D . Purification, biochemical characterization and protein-DNA interactions of the I-CreI endonuclease produced in Escherichia coli. Nucleic Acids Res. 1997; 25(19):3767-76. PMC: 146977. DOI: 10.1093/nar/25.19.3767. View

Nagai K, Thogersen H . Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 1987; 153:461-81. DOI: 10.1016/0076-6879(87)53072-5. View

Colleaux L, dAuriol L, Galibert F, Dujon B . Recognition and cleavage site of the intron-encoded omega transposase. Proc Natl Acad Sci U S A. 1988; 85(16):6022-6. PMC: 281897. DOI: 10.1073/pnas.85.16.6022. View

Wenzlau J, Saldanha R, Butow R, Perlman P . A latent intron-encoded maturase is also an endonuclease needed for intron mobility. Cell. 1989; 56(3):421-30. DOI: 10.1016/0092-8674(89)90245-6. View

Delahodde A, Goguel V, Becam A, Creusot F, Perea J, Banroques J . Site-specific DNA endonuclease and RNA maturase activities of two homologous intron-encoded proteins from yeast mitochondria. Cell. 1989; 56(3):431-41. DOI: 10.1016/0092-8674(89)90246-8. View

Dujon B . Group I introns as mobile genetic elements: facts and mechanistic speculations--a review. Gene. 1989; 82(1):91-114. DOI: 10.1016/0378-1119(89)90034-6. View

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; 18(6):1407-13. PMC: 330504. DOI: 10.1093/nar/18.6.1407. View

Wada K, Wada Y, Doi H, Ishibashi F, Gojobori T, Ikemura T . Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res. 1991; 19 Suppl:1981-6. PMC: 331342. DOI: 10.1093/nar/19.suppl.1981. View

10.

Flick K, Jurica M, Monnat Jr R, Stoddard B . DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI. Nature. 1998; 394(6688):96-101. DOI: 10.1038/27952. View

11.

Silva G, Dalgaard J, Belfort M, Van Roey P . Crystal structure of the thermostable archaeal intron-encoded endonuclease I-DmoI. J Mol Biol. 1999; 286(4):1123-36. DOI: 10.1006/jmbi.1998.2519. View

12.

Kowalski J, Belfort M, Stapleton M, Holpert M, Dansereau J, Pietrokovski S . Configuration of the catalytic GIY-YIG domain of intron endonuclease I-TevI: coincidence of computational and molecular findings. Nucleic Acids Res. 1999; 27(10):2115-25. PMC: 148431. DOI: 10.1093/nar/27.10.2115. View

13.

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; 104(2):241-5. DOI: 10.1016/0378-1119(91)90256-b. View

14.

GIMBLE F, Thorner J . Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae. Nature. 1992; 357(6376):301-6. DOI: 10.1038/357301a0. View

15.

Durrenberger F, Rochaix J . 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. 1993; 236(2-3):409-14. DOI: 10.1007/BF00277141. View

16.

PERRIN A, Buckle M, Dujon B . Asymmetrical recognition and activity of the I-SceI endonuclease on its site and on intron-exon junctions. EMBO J. 1993; 12(7):2939-47. PMC: 413549. DOI: 10.1002/j.1460-2075.1993.tb05956.x. View

17.

Lambowitz A, Belfort M . Introns as mobile genetic elements. Annu Rev Biochem. 1993; 62:587-622. DOI: 10.1146/annurev.bi.62.070193.003103. View

18.

Duguid J, Bloomfield V, Benevides J, Thomas Jr G . Raman spectroscopy of DNA-metal complexes. I. Interactions and conformational effects of the divalent cations: Mg, Ca, Sr, Ba, Mn, Co, Ni, Cu, Pd, and Cd. Biophys J. 1993; 65(5):1916-28. PMC: 1225927. DOI: 10.1016/S0006-3495(93)81263-3. View

19.

Garrett R . DNA substrate specificity and cleavage kinetics of an archaeal homing-type endonuclease from Pyrobaculum organotrophum. Nucleic Acids Res. 1994; 22(22):4583-90. PMC: 308504. DOI: 10.1093/nar/22.22.4583. View

20.

Turmel M, Mercier J, Cote V, Otis C, Lemieux C . The site-specific DNA endonuclease encoded by a group I intron in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene introduces a single-strand break at low concentrations of Mg2+. Nucleic Acids Res. 1995; 23(13):2519-25. PMC: 307060. DOI: 10.1093/nar/23.13.2519. View