STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE HOMING ENDONUCLEASE PI-SCEIBY LIMITED PROTEOLYTIC CLEAVAGE AND MOLECULAR-CLONING OF PARTIAL DIGESTION PRODUCTS

PI-SceI is a member of an unusual class of rare cutting homing endonuc leases produced by an autocatalytic protein splicing from a precursor. To analyze the structural and functional domain organization of the e ndonuclease PI-SceI and to examine whether the DNA binding activity ca n 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 th e protein in the same region, Each fragment contains one of the two co nserved LAGLIDADG motifs. The products of the limited proteolytic dige sts were shown to remain associated and to exhibit specific DNA bindin g 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 electro phoretic mobility shift assay. Expression clones for the N- and C-term inal 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 li mited tryptic digest show that a DNA substrate with the full length re cognition sequence is bent by 45 degrees. This degree of bending is al so observed with a DNA containing only the right side of the recogniti on sequence, corresponding to one of the DNA cleavage products of PI-S ceI. 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 specifica lly and to induce one of the distortions observed to occur in the proc ess 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.