Resolution of the EcoRII restriction endonuclease-DNA complex structure in solution using fluorescence spectroscopy

Abstract

The X-ray structure for the type IIE EcoRII restriction endonuclease has been resolved [X.E. Zhou, Y. Wang, M. Reuter, M. Mucke, D.H. Kruger, E.J. Meehan and L. Chen. Crystal structure of type IIE restriction endonuclease EcoRII reveals an autoinhibition mechanism by a novel effector-binding fold. J. Mol. Biol. 335 (2004) 307–319.], but the structure of the R.EcoRII–DNA complex is still unknown. The aim of this article was to examine the structure of the pre-reactive R.EcoRII–DNA complex in solution by fluorescence spectroscopy. The structure for the R.EcoRII–DNA complex was resolved by determining the fluorescence resonance energy transfer (FRET) between two fluorescent dyes, covalently attached near the EcoRII recognition sites, that were located at opposite ends of a lengthy two-site DNA molecule. Analysis of the FRET data from the two-site DNA revealed a likely model for the arrangement of the two EcoRII recognition sites relative to each other in the R.EcoRII–DNA complex in the presence of Ca 2+ ions. According to this model, the R.EcoRII binds the two-site DNA and forms a DNA loop in which the EcoRII recognition sites are 20 ± 10 Å distant to each other and situated at an angle of 70 ± 10°.