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 Mg~(2+)

摘要

Two group I introns (CpSSU centre dot 1 and CpSSU centre dot 2) that each potentially encode a protein with two copies of the LAGU-DADG motif were identified in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene. They both belongto subgroup IA3 and represent novel insertion positions in this gene (sites 508 and 793 in the Escherlchla coll 16S rRNA). The proteins encoded by the two introns were synthesized In vitro and tested for their ability to cleave the homing site of their respective Introns. Only the CpSSU 1-encoded protein (l-Cpall) was found to display specific DNA endonuclease activity. At 0.1 mM MgCI_2, l-Cpall nicks only the bottom (transcribed) DNA strand, but at concentrations ranging from 0.5 to 5.0 mM, it cleavesboth DNA strands (leaving a 4 nucleotide single-stranded extension with 3'-OH overhangs) while preferentially nicking the bottom strand. The rate of cleavage of the top strand increases with increasing concentration of MgCI_2. The preliminary data derived from these endonuclease assays suggest that the mode of DNA cleavage by l-Cpall is directed by the availability of Mg~(2+) and the affinity of different binding sites for this cation.