title: Structural and functional analysis of the homing endonuclease PI-sceI by limited proteolytic cleavage and molecular cloning of partial digestion products.
abstract: PI-SceI is a member of an unusual class of rare cutting homing endonucleases produced by an autocatalytic protein splicing from a precursor. To analyze the structural and functional domain organization of the endonuclease PI-SceI and to examine whether the DNA binding activity can be structurally separated from the catalytic activity, we performed limited proteolytic digestion experiments with various proteases. Two protease-resistant fragments spanning the N- and C-terminal halves of the nuclease were identified using different proteases which cleave the protein in the same region. Each fragment contains one of the two conserved LAGLIDADG motifs. The products of the limited proteolytic digests were shown to remain associated and to exhibit specific DNA binding but to be inactive in DNA cleavage. Different from what is observed with native PI-SceI, only one complex is formed as shown in an electrophoretic mobility shift assay. Expression clones for the N- and C-terminal protein fragments obtained by tryptic digestion were constructed, and the proteins PI-SceI-N and PI-SceI-C were purified. Only PI-SceI-N exhibits DNA binding activity. Bending experiments with PI-SceI-N, a mixture of PI-SceI-N and PI-SceI-C, as well as the products of the limited tryptic digest show that a DNA substrate with the full length recognition sequence is bent by 45;. This degree of bending is also observed with a DNA containing only the right side of the recognition sequence, corresponding to one of the DNA cleavage products of PI-SceI. Our results demonstrate that the N-terminal half of PI-SceI which lacks one of the two LAGLDADG motifs is able to bind to DNA specifically and to induce one of the distortions observed to occur in the process of DNA binding by PI-SceI. These results are discussed in light of the recently solved crystal structure of PI-SceI and used to refine a model for the mechanism of DNA binding and cleavage by PI-SceI.
authors.affiliation: Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Giessen, Germany. vera.pingoud@chemie.bio.uni-giessen.de
authors.forename: V
authors.initials: V
authors.lastname: Pingoud
authors.forename: W
authors.initials: W
authors.lastname: Grindl
authors.forename: W
authors.initials: W
authors.lastname: Wende
authors.forename: H
authors.initials: H
authors.lastname: Thole
authors.forename: A
authors.initials: A
authors.lastname: Pingoud
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002918:Chymotrypsin; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019286:Endopeptidase K; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering; D006180:Proton-Translocating ATPases; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D013381:Subtilisins; D014357:Trypsin
chemical_list: D004271:DNA, Fungal; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin; D019286:Endopeptidase K; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae