DNA substrate specificity and cleavage kinetics of an archaeal homing-type endonuclease from Pyrobaculum organotrophum.
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
J
Institute of Molecular Biology, Copenhagen University, Denmark.
Thi-Ngoc
H P
HP
Garrett
R A
RA
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D001482:Base Composition; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007438:Introns / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D003854:Deoxyribonucleotides; D012338:RNA, Ribosomal, 23S; D003850:Deoxyribonuclease I