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Amino Acid Residues in Both the Protein Splicing and Endonuclease Domains of the PI-SceI Intein Mediate DNA Binding

1998; Elsevier BV; Volume: 273; Issue: 8 Linguagem: Inglês

10.1074/jbc.273.8.4607

1083-351X

Zening He, Michael Crist, Hsiao-ching Yen, Xiaoqun Duan, Florante A. Quiocho, Frederick S. Gimble,

Viral Infections and Immunology Research

A structure-based model describing the interaction of the two-domain PI- Sce I 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) interacts with a distal region that is sufficient for high affinity binding. To support this model, alanine-scanning mutagenesis was used to assemble a set of 49 PI- Sce I mutant proteins that were purified and assayed for their DNA binding and cleavage properties. Fourteen mutant proteins were 4- to >500-fold less active than wild-type PI- Sce I in cleavage assays, and one mutant (T225A) was 3-fold more active. Alanine substitution at two positions in domain I reduces overall binding >60-fold by perturbing the interaction of PI- Sce I with the minimal binding region. Conversely, mutations in domain II have little effect on binding, reduce binding to the cleavage site region only, or affect binding to both regions. Interestingly, substitutions at Lys 301 , which is part of the endonucleolytic active site, eliminate binding to the cleavage site region but permit contact with the minimal binding region. This experimental evidence demonstrates that the protein splicing domain as well as the endonuclease domain is involved in binding of a DNA substrate with the requisite length.