title: Crosslinking the EcoRV restriction endonuclease across the DNA-binding site reveals transient intermediates and conformational changes of the enzyme during DNA binding and catalytic turnover.
abstract: EcoRV completely encircles bound DNA with two loops, forming the entry and exit gate for the DNA substrate. These loops were crosslinked generating CL-EcoRV which binds and releases linear DNA only slowly, because threading linear DNA into and out of the DNA-binding 'tunnel' of CL-EcoRV is not very effective. If the crosslinking reaction is carried out with a circular bound DNA, CL-EcoRV is hyperactive towards the trapped substrate which is cleaved very quickly but not very accurately. CL-EcoRV also binds to, but does not cleave, circular DNA when added from the outside, because it cannot enter the active site. Based on these results a two-step binding model is proposed for EcoRV: initial DNA binding occurs at the outer side of the loops before the gate opens and then the DNA is transferred to the catalytic center.
authors.affiliation: Institut für Biochemie (Fachbereich Biologie), Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen.
authors.forename: C
authors.initials: C
authors.lastname: Schulze
authors.forename: A
authors.initials: A
authors.lastname: Jeltsch
authors.forename: I
authors.initials: I
authors.lastname: Franke
authors.forename: C
authors.initials: C
authors.lastname: Urbanke
authors.forename: A
authors.initials: A
authors.lastname: Pingoud
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers; D004270:DNA, Circular / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D004270:DNA, Circular; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases