Implications for Switching Restriction Enzyme Specificities from the Structure of BstYI Bound to a BglII DNA Sequence

Abstract

The type II restriction endonuclease BstYI recognizes the degenerate sequence 5'-RGATCY-3' (where R = A/G and Y = C/T), which overlaps with both BamHI (GGATCC) and BglII (AGATCT), and thus raises the question of whether BstYI DNA recognition will be more BamHI-like or BglII-like. We present here the structure of BstYI bound to a cognate DNA sequence (AGATCT). We find the complex to be more BglII-like with similarities mapping to DNA conformation, domain organization, and residues involved in catalysis. However, BstYI is unique in containing an extended arm subdomain, and the mechanism of DNA capture has both BglII-like and BamHI-like elements. Further, DNA recognition is more minimal than BglII and BamHI, where only two residues mediate recognition of the entire core sequence. Taken together, the structure reveals a mechanism of degenerate DNA recognition and offers insights into the possibilities and limitations in altering specificities of closely related restriction enzymes.

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

“…However, solution studies are still needed to confirm the structural interpretation of metal ion coordination by BamHI and BglII". 52 We decided to address this problem experimentally by studying the Mg 2+ and Mn 2+ concentration dependence of DNA cleavage by representative type II restriction endonucleases of the EcoRI family: BamHI, BglII, Cfr10I, EcoRI, EcoRII-C, MboI, Ngo-MIV, PspGI, and SsoII. Members of this family have been shown to have one or two divalent metal ions per active site in the co-crystal structure of the respective enzyme-DNA complex.…”

Early Interrogation and Recognition of DNA Sequence by Indirect Readout

“…Secondly, this indirect readout can also be used during target site location by an intermediate in the pathway having limited DNA contacts and distortions. Thirdly, enzymes recognizing similar yet distinct sequences may distort DNA differently to prevent specificity conversion by one or two amino acid substitutions (Townson et al, 2005). Finally, new specificities cannot evolve by merely substituting one amino acid for another at the site of the direct contacts, since the side chains are generally not isosteric.…”

Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity

“…Subsequently the structure of BstYI was determined (14). Analysis of the structures revealed that although BstYI, BglII and BamHI share many similarities and are ideal test cases for changing specificity, the enzymes use surprisingly distinct recognition strategies, leaving open the question whether it is possible to completely switch specificity, even for the relatively conservative change of reducing BstYI recognition to specificity for only the BglII sequence (15). In another study, an approach that used random mutagenesis coupled with a genetic screen was employed in an attempt to alter the specificity of NotI (GCGGCCGC).…”