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DNA substrate specificity and cleavage k ... from Pyrobaculum organotrophum
DNA substrate specificity and cleavage kinetics of an archaeal homing-type endonuclease from Pyrobaculum organotrophum.
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The protein encoded by intron 1 of the single 23S rRNA gene of the archaeal hyperthermophile Pyrobaculum organotrophum was isolated and shown to constitute a homing-type DNA endonuclease, I-PorI. It cleaves the intron- 23S rDNA of the closely related organism Pyrobaculum islandicum near the site of intron insertion in Pb.organotrophum. In contrast, no endonuclease activity was detected for the protein product of intron 2 of the same gene of Pb.organotrophum which, like I-PorI, carries the LAGLI-DADG motif, common to group I intron-encoded homing enzymes. I-PorI cleaves optimally at 80 degrees C, with kcat and Km values of about 2 min-1 and 4 nM, respectively, and generates four nucleotide 3'-overhangs and 5'-phosphates. It can cleave a 25 base pair DNA fragment encompassing the intron insertion site. A mutation-selection study established the base pair specificity of the endonuclease within a 17 bp region, from positions -6 to +11 with respect to the intron-insertion site. Four of the essential base pairs encode the sequence involved in the intron-exon interaction in the pre-rRNA that is required for recognition by the RNA splicing enzymes. Properties of the enzyme are compared and contrasted with those of eucaryotic homing endonucleases.
Lykke-Andersen J, Thi-Ngoc HP, Garrett RA
Nucleic acids research
1994-11-11 00:00
22
22
4583-90
Amino Acid Sequence,Archaea,Base Composition,Base Sequence,DNA, Bacterial,DNA, Ribosomal,Deoxyribonuclease I,Deoxyribonucleotides,Escherichia coli,Gene Expression,Genetic Vectors,Introns,Kinetics,Molecular Sequence Data,Point Mutation,RNA, Ribosomal, 23S,Substrate Specificity,Temperature,DNA, Bacterial,DNA, Ribosomal,Deoxyribonucleotides,RNA, Ribosomal, 23S,Deoxyribonuclease I
Institute of Molecular Biology, Copenhagen University, Denmark
Nucleic Acids Res.
0305-1048
804
True
7984405
