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

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 l inear 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 boun d DNA, CL-EcoRV is hyperactive towards the trapped substrate which is cleav ed very quickly but not very accurately. CL-EcoRV also binds to, but does n ot cleave, circular DNA when added from the outside, because it cannot ente r the active site. Based on these results a two-step binding model is propo sed for EcoRV: initial DNA binding occurs at the outer side of the loops be fore the gate opens and then the DNA is transferred to the catalytic center .