Glucocorticoid Receptor-like Zn(Cys)4 Motifs in BslI Restriction Endonuclease

BslI restriction endonuclease cleaves the symmetric sequence CCN7GG (where N=A, C, G or T). The enzyme is composed of two subunits, α and β, that form a heterotetramer (α2β2) in solution. The α subunit is believed to be responsible for DNA recognition, while the β subunit is thought to mediate cleavage. Here, for the first time, we provide experimental evidence that BslI binds Zn(II). Specifically, using X-ray absorption spectroscopic analysis we show that the α subunit of BslI contains two Zn(Cys)4-type zinc motifs similar to those in the DNA-binding domain of the glucocorticoid receptor. This conclusion is supported by genetic analysis of the zinc-binding motifs, whereby amino acid substitutions in the zinc finger motifs are demonstrated to abolish or impair cleavage activity. An additional putative zinc-binding motif was identified in the β subunit, consistent with the X-ray absorption data. These data were corroborated by proton induced X-ray emission measurements showing that full BslI contains at least three fully occupied Zn sites per α/β heterodimer. On the basis of these data, we propose a role for the BslI Zn motifs in protein–DNA as well as protein–protein interactions.