Cleavage and recognition pattern of a double-strand-specific endonuclease (I-creI) encoded by the chloroplast 23S rRNA intron of Chlamydomonas reinhardtii.
Several group-I introns have been shown to specifically invade intron-minus alleles of the genes that contain them. This type of intron mobility is referred to as 'intron homing', and depends on restriction endonucleases (ENases) encoded by the mobile introns. The ENase cleaves the intron-minus allele near the site of intron insertion, thereby initiating gene conversion. The 23S (LSU) rRNA-encoding gene (LSU) of the chloroplast genome of Chlamydomonas reinhardtii contains a self-splicing group-I intron (CrLSU) that has a free-standing open reading frame (ORF) of 163 codons. Translation of CrLSU intron RNA in cell-free systems produces a polypeptide of approx. 18 kDa, the size expected for correct translation of the ORF. The in vitro-synthesized 18-kDa protein cleaves plasmid DNA that contains a portion of LSU where the intron normally resides, but lacking the intron itself. Cleavage by the intron-encoded enzyme (I-CreI) occurs 5 bp and 1 bp 3' to the intron insertion site (in the 3'-exon) in the top (/) and bottom (,) strands, respectively, resulting in 4-nt single-stranded overhangs with 3'-OH termini. We also show that the recognition sequence of I-CreI spans the cleavage site and is 24 bp in length (5'-CAAAACGTC,GTGA/GACAGTTTGGT).
Thompson
A J
AJ
Department of Botany, University of Texas, Austin 78713.
Yuan
X
X
Kudlicki
W
W
Herrin
D L
DL
D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology*; D004247:DNA; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D007438:Introns; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D015183:Restriction Mapping; D013379:Substrate Specificity
D012338:RNA, Ribosomal, 23S; D004247:DNA; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI; D015252:Deoxyribonucleases, Type II Site-Specific