AMINO-ACID-RESIDUES IN BOTH THE PROTEIN SPLICING AND ENDONUCLEASE DOMAINS OF THE PI-SCEI INTEIN MEDIATE DNA-BINDING

A structure-based model describing the interaction of the two-domain P I-SceI endonuclease with its 31-base pair DNA substrate suggests that the endonuclease domain (domain II) contacts the cleavage site region of the substrate, while the protein splicing domain (domain I) interac ts with a distal region that is sufficient for high affinity binding, To support this model, alanine-scanning mutagenesis was used to assemb le a set of 49 PI-SceI mutant proteins that were purified and assayed for their DNA binding and cleavage properties, Fourteen mutant protein s were 4- to >500-fold less active than wild-type PI-SceI in cleavage assays, and one mutant (T225A) was 3-fold more active, Alanine substit ution at two positions in domain I reduces overall binding >60-fold by perturbing the interaction of PI-SceI with the minimal binding region . Conversely, mutations in domain II have little effect on binding, re duce binding to the cleavage site region only, or affect binding to bo th regions, Interestingly, substitutions at Lys(301), which is part of the endonucleolytic active site, eliminate binding to the cleavage si te region but permit contact with the minimal binding region, This exp erimental evidence demonstrates that the protein splicing domain as we ll as the endonuclease domain is involved in binding of a DNA substrat e with the requisite length.