Structural and energetic origins of indirect readout in the recognition of GATATC by EcoRV endonuclease

EcoRV endonuclease has extreme specificity for its recognition sequence GATATC. It must discriminate against the vast number of similar substrates which comprise the rest of the bacterial genome. The cognate co-crystal structure shows numerous specific protein-DNA interactions which account in large measure for this specificity. However, greater than 50% of the specificity at the center TA step is achieved through “indirect readout”. The enzyme sharply bends the DNA by 50° at the center step. This distorted conformation is significantly less energetically favorable for non-cognate sequences than for the cognate sequence. The specific contacts at the center TA step are important in positioning the scissile phosphate for catalysis, in achieving the correct protein conformation, in forming required divalent metal binding sites, and in sequence specificity.; The sequence GAATTC cannot achieve the distorted cognate conformation due to a steric same-strand clash within the DNA. The DNA in this noncognate complex is bent by only 30° at the center step, significantly less than in the cognate complex. Removal of a methyl group involved in the clash reconstitutes the severe bending of the DNA in the complex (49°).; Discrimination against the sequences GACGTC and GAGCTC is more subtle. These sequences can achieve the distorted conformation seen in the cognate crystal structure. However, the dinucleotide steps CG and GC have very favorable base stacking energies, while the TA step has very little stacking energy. The distortion of CG and GC is significantly less energetically favorable than is the distortion of TA.; Mg2+ is also an important factor in the specificity of EcoRV endonuclease for its substrate. The presence of divalent metal at the protein-DNA interface serves to relieve the charge repulsion between the acidic metal-ligating protein residues and the negatively charged DNA phoshate backbone.; Additionally, the entire course of the EcoRV endonuclease reaction has been monitored by fluorescence anisotropy and energy transfer assays. Binding and bending are simultaneous for the cognate substrate. This is the first demonstration of simultaneous DNA binding and bending by an enzyme. Bending becomes two-fold slower than binding for non-cognate substrates.