Cleavage pattern of the homing endonuclease encoded by the fifth intron in the chloroplast large subunit rRNA-encoding gene of Chlamydomonas eugametos.

The fifth group-I intron in the chloroplast large subunit rRNA-encoding gene of Chlamydomonas eugametos (CeLSU.5) is mobile during interspecific crosses between C. eugametos and Chlamydomonas moewusii. Like the six other mobile introns that have been well characterized so far, CeLSU.5 contains a long open reading frame (ceuIR) coding for a site-specific endonuclease (I-CeuI) that cleaves the C. moewusii intronless gene in the vicinity of the intron-insertion site. This stimulates gap repair and mediates efficient transfer of the intron at its cognate site. By expressing the ceuIR gene in the Escherichia coli vectors pKK233-2 and pTRC-99A, we recently demonstrated that the endonuclease is highly toxic to E. coli [Gauthier et al., Curr. Genet. 19 (1991) 43-47]. To eliminate this problem and characterize the cleavage pattern and recognition sequence of the I-CeuI endonuclease, we have expressed the ceuIR gene in E. coli under the control of a bacteriophage T7 promoter in a tightly regulated M13 system, and developed an in vitro system to assay partially purified I-CeuI activity. This allowed us to determine that I-CeuI recognizes a sequence of less than 26 bp centered around the insertion site and produces a staggered cut 5 bp downstream from this site, yielding 4-nucleotide (CTAA), 3'-OH overhangs.