ACCURACY OF THE ECORV RESTRICTION-ENDONUCLEASE - BINDING AND CLEAVAGESTUDIES WITH OLIGODEOXYNUCLEOTIDE SUBSTRATES CONTAINING DEGENERATE RECOGNITION SEQUENCES

In order to investigate the accuracy of the EcoRV restriction endonucl ease, we have synthesized a set of double-stranded oligodeoxynucleotid es comprising the canonical recognition sequence, the 9 star sequences (i.e., sequences deviating by one base pair from the canonical sequen ce), and the 18 mismatch sequences (i.e. sequences deviating by one ba se from the canonical sequence). For each individual single strand of all these 28 substrates we have measured the rate of phosphodiester bo nd cleavage under normal buffer conditions. Double-strand cleavage of star substrates is at least 5 orders of magnitude slower than cleavage of the canonical substrate. In contrast, most of the mismatch substra tes are accepted more readily. In the absence of the essential cofacto r Mg2+, EcoRV binds weakly but equally to the canonical and degenerate substrates, (i.e., K-Diss is in the micromolar range). However, the i nactive catalytic site mutant D90A in the presence of Mg2+ binds the c anonical substrate 1-2 orders of magnitude better than degenerate subs trates. Therefore, the EcoRV endonuclease needs the essential cofactor Mg2+ to create thermodynamic discrimination between degenerate and ca nonical sites. But the main discrimination is kinetically controlled a nd takes place during cleavage. While in the canonical substrate both single strands are cleaved with an equal velocity, in all other substr ates one single strand is cleaved faster than the other one, resulting in a dissociation of the enzyme from the DNA between the two cuts. In vivo this may lead to a repair of the erroneous cleavage site by DNA ligases. The order of single-strand nicking together with the division of base contacts on both subunits suggests that correct recognition b y one subunit triggers cleavage by the other one.