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The activities of enzymes with mutations in the beta-bridge segment are studied. The results reveal that the enzyme recognizes its substrates by DNA conformation exclusion and offer a simple explanation for the broad substrate specificity of phage resolvase.

Restriction endonuclease BglII completely encircles its target DNA, making contacts to both the major and minor grooves. To allow the DNA to enter and leave the binding cleft, the enzyme dimer has to rearrange. To understand how this occurs, we have solved the structure of the free enzyme at 2.3 Å resolution, as a complement to our earlier work on the BglII?DNA complex. Unexpectedly, the enzyme opens by a dramatic `scissor-like' motion, accompanied by a complete rearrangement of the α-helices at the dimer interface. Moreover, within each monomer, a set of residues ? a ‘lever’ ? lowers or raises to alternately sequester or expose the active site residues. Such an extreme difference in free versus complexed structures has not been reported for other restriction endonucleases. This elegant mechanism for capturing DNA may extend to other enzymes that encircle DNA. [ABSTRACT FROM AUTHOR]

The MspI restriction endonuclease is a type II restriction enzyme. Unlike all other restriction enzymes with known structures, MspI recognizes the palindromic tetranucleotide sequence 5′-C/CGG and cleaves it as indicated by the `I' to produce DNA products with 5' two- base overhangs. Owing to the nature of its cleavage pattern, it is likely that MspI would represent a new structural class of restriction endonucleases. Crystals of the dimeric MspI restriction enzyme bound to a duplex DNA molecule containing the specific recognition sequence have been obtained by vapor-diffusion techniques in the presence of polyethylene glycol as precipitant. The crystals belong to the monoclinic space group P21, with unit-cell parameters a 50.2, b = 131.6, c = 59.3 Å, β = 109.7°. The crystals contain one dimeric complex in the asymmetric unit. A complete native data set has been collected to a resolution of 2.05 A by cryo-crystallographic methods, with an Rmerge of 4.0%. [ABSTRACT FROM AUTHOR]

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1.5 Å resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar α/β core, but in BglII, the core is augmented by a β-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein?DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases. [ABSTRACT FROM AUTHOR]