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

The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGL IDADG 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-Scal promoting intron and as a maturase promoting in tron Here we describe the synthesis of a universal code equivalent to the m itochondrial sequence coding for this protein and the in vitro characteriza tion of I-Scal endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli, We have also determined the cl eavage pattern as well as the recognition site of p28bi2. It was found that p28bi2 generates a double-strand cleavage downstream from the intron inser tion site with 4 nt long 3'-overhangs. Mutational analysis of the DNA targe t site shows that p28bi2 recognizes a 16-19 bp sequence from positions -11 to +8 with respect to the intron insertion site.