THE DESIGNED PROTEIN M(II)-GLY-LYS-HIS-FOS(138-211) SPECIFICALLY CLEAVES THE AP-1 BINDING-SITE CONTAINING DNA

A new specific DNA cleavage protein, Gly-Lys-His-Fos(138-211), was des igned, expressed, and characterized. The DNA-binding component of the design uses the basic and leucine zipper regions of the leucine zipper Fos, which are represented by Fos(138-211). The DNA cleavage moiety w as provided by the design of the amino-terminal Cu(II)-, Ni(II)-bindin g site GKH at the amino terminus of Fos(138-211). Binding of Cu(II) or Ni(LI) by the protein activates its cleavage ability. The GKH motif w as predicted to form a specific amino-terminal Cu(II)-, Ni(LT)-binding motif as previously defined [Predki, P. F., Harford, C., Brar, P., & Sarkar, B. (1992) Biochem. J. 287, 211-215]. This prediction was verif ied as the tripeptide, GKH, and the expressed protein, GKH-Fos(138-211 ), were both shown to be capable of binding Cu(II) and Ni(II). The des igned protein upon heterodimerization with Jun(248-334) was shown to b ind to and cleave several forms of DNA which contained an AP-I binding site. The cleavage was shown to be specific. This design demonstrates the versatility of the amino-terminal Cu(II)-, Ni(II)-binding motif a nd the variety of motifs which can be generated. The site of cleavage by GKH-Fos(138-211) on DNA provides further information regarding the bending of DNA upon binding to Fos-Jun heterodimers.