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 Mg2+.
Two group I introns (CpSSU.1 and CpSSU.2) that each potentially encode a protein with two copies of the LAGLI-DADG motif were identified in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene. They both belong to subgroup IA3 and represent novel insertion positions in this gene (sites 508 and 793 in the Escherichia coli 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 (I-CpaII) was found to display specific DNA endonuclease activity. At 0.1 mM MgCl2, I-CpaII nicks only the bottom (transcribed) DNA strand, but at concentrations ranging from 0.5 to 5.0 mM, it cleaves both 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 MgCl2. The preliminary data derived from these endonuclease assays suggest that the mode of DNA cleavage by I-CpaII is directed by the availability of Mg2+ and the affinity of different binding sites for this cation.
Turmel
M
M
Canadian Institute for Advanced Research, Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Canada.
Mercier
J P
JP
Côté
V
V
Otis
C
C
Lemieux
C
C
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002696:Chlamydomonas / Q000201:enzymology / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D007438:Introns; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012335:RNA, Ribosomal / Q000235:genetics*; D013379:Substrate Specificity
D012335:RNA, Ribosomal; D004247:DNA; D004706:Endodeoxyribonucleases; C094925:endodeoxyribonuclease I-CpaII; D008274:Magnesium