Mechanism of DNA Recognition by EcoRV

Abstract

The restriction enzyme EcoRV destroys invading foreign DNA by inducing a sharp kink of 50 degrees and cleaving it at the center step of a GATATC sequence. It's own DNA is methylated in the first adenine, GACH3TATC, and is not cleaved by EcoRV. We report here on molecular dynamics simulations of the interaction of EcoRV with three DNA sequences: the cognate sequence, GATATC, the cognate methylated sequence, GACH3TATC and the non-cognate sequence, GAATTC, not cleaved by EcoRV. Simulations of the three DNA sequences unbound and bound to EcoRV and of unbound EcoRV are performed, to understand the recognition-cleavage process. The results suggest a three-step recognition mechanism: first, EcoRV is in an open state, ready to bind to the DNA. When bound, EcoRV makes loose contacts with any DNA sequence. Then in a third step, taking place only for the cognate sequence, the DNA is kinked and bound deep enough in the protein to allow cleavage. This step is determined by an intrinsically higher flexibility of the cognate sequence and the formation of stronger hydrogen-bond interactions between DNA and protein than for the other two non-cleaved sequences. A crucial role of Asn185, forming hydrogen bonds with the first adenine of the recognition sequence, GATATC, could be determined from our simulations. In the EcoRV-methyl-DNA complex, as well as in the complex of a N185A mutant with the cognate sequence (TA), the formation of a hydrogen bond between Asn185 and the adenine is prohibited. The formation of a tight EcoRV-DNA complex is thus impossible and the energy gained upon complex formation becomes insufficient to kink the DNA despite its intrinsic flexibility. These findings elucidate in atomic detail the interplay between specific binding interactions in the complex and intrinsic properties of the DNA in the recognition process.