In the Arms of EcoRI - probing the Binding Specificity of the Restriction Endonuclease Using Electron Spin Resonance

Abstract

Pulsed electron spin resonance (ESR) was used to probe the binding specificity of EcoRI, a restriction endonuclease that binds to and cleaves a six base pair sequence of DNA. EcoRI binds to the specific sequence GAATTC with an affinity that is 50,000-90,000-fold greater than that of a miscognate site that differs by only one base pair. Low binding affinity is also exhibited at non-specific binding sites which differ from the specific sequence by two or more base pairs. Distance measurements were performed on several spin labeled EcoRI mutants when bound to specific, miscognate, and non-specific sequences of DNA using Double Electron-Electron Resonance. These distances demonstrated that on average the arms of EcoRI, thought to play a major role in binding specificity, are similarly positioned. Additionally, noncognate (miscognate and non-specific) complexes demonstrated broader distance distributions indicating that the flexibility of the arms is greater in these complexes. Room temperature continuous wave (CW) experiments were also performed on the EcoRI mutant complexes at both X-band and W-band to probe the arm region dynamics. Higher sensitivity to the fast motional dynamics of the spin label at W-band resolved differences in two of the EcoRI complexes that were not apparent in the X-band CW spectra. Molecular dynamics (MD) simulations were performed on the spin-label-modified specific EcoRI-DNA crystal structure to model the average nitroxide orientation. Disparity in average distance as well as distribution indicates a need for further sampling of the spin label in silico. This work is supported by NSF.