The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.

  title={The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.},
  author={F. Winkler and D. Banner and C. Oefner and D. Tsernoglou and R. Brown and S. Heathman and R. Bryan and P. Martin and K. Petratos and K. Wilson},
  journal={The EMBO Journal},
The crystal structure of EcoRV endonuclease has been determined at 2.5 A resolution and that of its complexes with the cognate DNA decamer GGGATATCCC (recognition sequence underlined) and the non-cognate DNA octamer CGAGCTCG at 3.0 A resolution. Two octamer duplexes of the non-cognate DNA, stacked end-to-end, are bound to the dimeric enzyme in B-DNA-like conformations. The protein--DNA interactions of this complex are prototypic for non-specific DNA binding. In contrast, only one cognate… Expand
Non-cognate enzyme-DNA complex: structural and kinetic analysis of EcoRV endonuclease bound to the EcoRI recognition site GAATTC.
The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity. Expand
Structure of HhaI endonuclease with cognate DNA at an atomic resolution of 1.0 Å
The structure of HhaI, a Type II restriction endonuclease, recognizes the symmetric sequence 5′-GCG↓C-3′ in duplex DNA and cleaves (‘↓’) to produce fragments with 2-base, 3′-overhangs, and is characterized in atomic detail. Expand
Role of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT) + d(ATCTT).
Results indicate that EcoRV bends non-cognate sites differing by one or two base-pairs from GATATC, but does not bend non-specific sites that are less similar, which suggests that the sequence-dependent energy cost of DNA bending is likely to play an important role in determining the specificity of Eco RV. Expand
Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis.
The D34G alteration results in several structural changes relative to wild-type protein/DNA complexes that imply that the Asp34 side-chains from the two subunits maintain a distinct conformation of its DNA substrate, properly situating the target backbone phosphates and indirectly manipulating the active sites. Expand
Restriction enzyme BsoBI-DNA complex: a tunnel for recognition of degenerate DNA sequences and potential histidine catalysis.
The structure of BsoBI complexed to cognate DNA has been determined to 1.7 A resolution, revealing several unprecedented features and providing two novel examples of the role of water in protein-DNA interaction. Expand
Structure–function correlation for the EcoRV restriction enzyme: from non‐specific binding to specific DNA cleavage
The EcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence because the rate of cleavage is controlled by the binding of Mg2+ to ecoRV‐DNA complexes: the complex at the recognition site has a high affinity for Mg 2+, while the complexes at other DNA sequences have low affinities for Mm2+. Expand
The energetics of the interaction of BamHI endonuclease with its recognition site GGATCC.
DNA binding is strongly enhanced by mutations D94N, E111A or E113K, by binding of Ca(2+) at the active site, or by deletion of the scissile phosphate GpGATCC, indicating that a cluster of negative charges at the catalytic site contributes at least 3-4 kcal/mol of unfavorable binding free energy. Expand
Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution.
The type II restriction endonuclease EcoRV was crystallized as a complex with the substrate DNA undecamer AAAGATATCTT and inspired by recent kinetic data, a transition state model with two metals bound to the scissile phosphorane group is constructed. Expand
Conformational changes and cleavage by the homing endonuclease I-PpoI: a critical role for a leucine residue in the active site.
Leucine 116 is critical for binding and catalysis: it appears to be important for forming a well-ordered protein-DNA complex at the cleavage site, for maximal deformation of the DNA, and for desolvation of the nucleotide bases that are partially unstacked in the enzyme complex. Expand
Catalytic efficiency and sequence selectivity of a restriction endonuclease modulated by a distal manganese ion binding site.
Characterization of the H71A and H71Q mutants of EcoRV demonstrates that these distal Mn(2+) sites significantly modulate activity toward both cognate and non-cognate DNA substrates, revealing a strong dependence of DNA cleavage efficiency upon metal ion-mediated interactions located some 20 A distant from the scissile phosphodiester linkages. Expand