PROTEIN ENGINEERING OF THE RESTRICTION-ENDONUCLEASE ECORV - STRUCTURE-GUIDED DESIGN OF ENZYME VARIANTS THAT RECOGNIZE THE BASE-PAIRS FLANKING THE RECOGNITION SITE

We generated variants of the restriction endonuclease EcoRV that discr iminate between recognition sites with different flanking sequences. T his was achieved by designing new contacts to the bases in the major g roove of the DNA preceding and following the EcoRV recognition site. W e selected Ala181 as the starting point for the extension of the site specificity of EcoRV because, according to the structure of the specif ic EcoRV DNA complex, this residue is involved in a water mediated con tact with the bases flanking the recognition sequence on the 5' side. A substitution of this alanine residue by other amino acid residues ch anges the protein-DNA interface in this region and potentially creates new contacts, such that EcoRV variants could have an extended specifi city, i.e. a greater selectivity for EcoRV sites within a particular s equence context. EcoRV variants with naturally occuring amino acid res idues at position 181 were produced and their selectivity analyzed wit h oligodeoxynucleotide and plasmid substrates that differ only in the base pairs immediately flanking the EcoRV site. Some variants, having amino acid residues with long or bulky side chains at position 181 sho wed altered preferences for the base pairs flanking the recognition se quence with oligodeoxynucleotide substrates without loosing their cata lytic efficiency. One variant, A181K, is able to discriminate between purine and pyrimidine bases on the 5' side of the recognition sequence , probably by means of a new hydrogen bond to the N7 of the purine bas e. Another variant, A181E, strongly prefers a thymine base on the 5' s ide of the recognition sequence, presumably due to a hydrogen bond for med between the protonated glutamic acid residue and the O4 of thymine .