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Nucleic Acids Research, 2000, Vol. 28, No. 5 1245-1251
© 2000 Oxford University Press

Purification and characterization of the DNA cleavage and recognition site of I-ScaI mitochondrial group I intron encoded endonuclease produced in Escherichia coli

Claude Monteilhet*, Dorota Dziadkowiec, Tomasz Szczepanek and Jaga Lazowska

Centre de Génétique Moléculaire CNRS, 91198 Gif-sur Yvette Cedex, France

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-ScaI 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-ScaI 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.

* To whom correspondence should be addressed. Tel: +33 1 69 82 31 87; Fax: +33 1 69 07 55 39; Email: monteilhet@cgm.cnrs-gif.fr


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