Glucocorticoid receptor-like Zn(Cys)4 motifs in BslI restriction endonuclease.
BslI restriction endonuclease cleaves the symmetric sequence CCN(7)GG (where N=A, C, G or T). The enzyme is composed of two subunits, alpha and beta, that form a heterotetramer (alpha(2)beta(2)) in solution. The alpha subunit is believed to be responsible for DNA recognition, while the beta 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 alpha 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 beta 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 alpha/beta 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.
Vanamee
Eva Scheuring
ES
Structural Biology Program, Department of Physiology and Biophysics, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA.
Hsieh
Pei chung
Pc
Zhu
Zhenyu
Z
Yates
David
D
Garman
Elspeth
E
Xu
Shuang yong
Sy
Aggarwal
Aneel K
AK
D001665:Binding Sites; D003596:Cytosine / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000737:chemistry; D014965:X-Rays; D015032:Zinc / Q000502:physiology*
D011965:Receptors, Glucocorticoid; D003596:Cytosine; D004247:DNA; C404107:endodeoxyribonuclease BslI; D015252:Deoxyribonucleases, Type II Site-Specific; D015032:Zinc