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. 1998 Nov 16;17(22):6757–6766. doi: 10.1093/emboj/17.22.6757

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.

C Schulze 1, A Jeltsch 1, I Franke 1, C Urbanke 1, A Pingoud 1
PMCID: PMC1171021  PMID: 9822618

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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