Activity and specificity of the bacterial PD-(D/E)XK homing endonuclease I-Ssp6803I.

Publication Type:

Journal Article


Journal of molecular biology, Volume 385, Issue 5, p.1498-510 (2009)


2009, Amino Acid Sequence, Bacterial Proteins, Basic Sciences Division, DNA Restriction Enzymes, DNA, Bacterial, Models, Molecular, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, Protein Binding, Protein Folding, Protein Structure, Quaternary, Protein Structure, Secondary, Synechocystis


The restriction endonuclease fold [a three-layer alpha-beta sandwich containing variations of the PD-(D/E)XK nuclease motif] has been greatly diversified during evolution, facilitating its use for many biological functions. Here we characterize DNA binding and cleavage by the PD-(D/E)XK homing endonuclease I-Ssp6803I. Unlike most restriction endonucleases harboring the same core fold, the specificity profile of this enzyme extends over a long (17 bp) target site. The DNA binding and cleavage specificity profiles of this enzyme were independently determined and found to be highly correlated. However, the DNA target sequence contains several positions where binding and cleavage activities are not tightly coupled: individual DNA base-pair substitutions at those positions that significantly decrease cleavage activity have minor effects on binding affinity. These changes in the DNA target sequence appear to correspond to substitutions that uniquely increase the free energy change between the ground state and the transition state, rather than simply decreasing the overall DNA binding affinity. The specificity of the enzyme reflects constraints on its host gene and limitations imposed by the enzyme's quaternary structure and illustrate the highly diverse repertoire of DNA recognition specificities that can be adopted by the related folds surrounding the PD-(D/E)XK nuclease motif.