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

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.