DNA substrate specificity and cleavage kinetics of an archaeal homing-type endonuclease from Pyrobaculum organotrophum.
AUTOR(ES)
Lykke-Andersen, J
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=308504Documentos Relacionados
- PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities.
- Mutations altering the cleavage specificity of a homing endonuclease
- Analysis of substrate specificity of the PaeR7 endonuclease: effect of base methylation on the kinetics of cleavage.
- Profile of the DNA recognition site of the archaeal homing endonuclease I-DmoI.
- Binding, bending and cleavage of DNA substrates by the homing endonuclease Pl-SceI.