Resolution of the EcoRII restriction endonuclease-DNA complex structure in solution using fluorescence spectroscopy.
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 A distant to each other and situated at an angle of 70+/-10 degrees.
Subach
Fedor
F
Department of Chemistry, Moscow State University, Moscow, 119991, Russia.
Kirsanova
Olga
O
Liquier
Jean
J
Gromova
Elizaveta S
ES
D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012996:Solutions
D012996:Solutions; D004247:DNA; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific