Abstract
A structure-based model describing the interaction of the two-domain PI-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) 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-SceI 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-SceI 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-SceI 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 Lys301, 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.
Highlights
The yeast PI-SceI endonuclease catalyzes the hydrolysis of two specific phosphodiester bonds within an asymmetrical recognition site [1]
Region I contains the cleavage site that is cut by the enzyme to generate a 4-base pair overhang, and region II includes an adjacent 17-bp sequence that is sufficient for high affinity binding
To determine the identity of amino acid residues involved in contacting the PI-SceI recognition sequence, we used alaninescanning mutagenesis to create a set of mutant proteins with single amino acid changes at numerous positions on the proposed DNA binding interface
Summary
Materials—TALON metal affinity resin and TALONspin columns were obtained from CLONTECH. Elution fractions containing PI-SceI, as judged by SDS-polyacrylamide gel electrophoresis, were pooled and dialyzed overnight in buffer D (10 mM potassium phosphate (pH 7.6), 5% glycerol, 0.1 mM EDTA, and 1.4 mM 2-mercaptoethanol) containing 40 mM KCl (buffer D40). In addition to wild-type PI-SceI, the R90A, R94A, T225A, R231A, D232A, Y328A, T338A, and H343A mutant proteins formed PDLC and PDUC complexes, and binding could be represented by Scheme I (Fig. 2). PI-SceI Cleavage Analysis—In an initial characterization of the PISceI proteins, purified enzyme (50 –150 nM) was incubated with XmnIlinearized pBS-PISce (7 nM) [4] in 15 l of cleavage buffer (100 mM KCl, 25 mM Tris-HCl (pH 8.5), 2.5 mM 2-mercaptoethanol, 2.5 mM MgCl2) for 30 min and 1 h at 37 °C. Cleavage rates were calculated from curve fitting of the linear portions of the reaction using KaleidaGraph (Synergy Software)
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