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.

Mechanism of DNA Recognition by the Restriction Enzyme EcoRV

“…Second, these data confirm the existence of an "open state" of unbound EcoRV in solution. This state has not been observed experimentally, but was, according to Schulze et al, 28 "never ruled out. "…”

“…28, it was noted that the radii of gyration of EcoRV free and in complex with specific DNA, as determined by neutron scattering, are identical within the limits of error. We have now computed the radius of gyration for the simulations of the protein free in the crystal, in solution and the protein complexed to DNA.…”

“…27 The existence of an open conformation was not ruled out, although there is no experimental evidence for an open state of free EcoRV in solution. 28 Crystal structures of EcoRV and of the cognate EcoRV-DNA complex ( Fig. 1a and b) reveal extensive conformational changes in both protein and DNA upon complex formation.…”

Type II restriction endonucleases: structure and mechanism

“…It is noteworthy that the H-N-H homing endonuclease I-CmoeI is also active with Ca 2+ [93]. [95]. All restriction endonucleases face the problem of efficiently finding their specific site in the presence of a huge excess of non-specific sites, to which they can also bind although with a much lower affinity [96].…”

“…Pingoud, unpublished]. The importance of sliding can be deduced from the finding that triple-helix formation over 16 bp interferes with efficient target site location [105] and from the fact that an EcoRV molecule whose DNA binding site has been closed by a cross-link after non-specific binding to a circular DNA molecule is perfectly able to find and cleave its specific site after addition of Mg 2+ [95]. In addition, during linear diffusion specific sites are not overlooked [105,109,115], which is most easily explained by sliding as a major determinant for facilitated diffusion.…”