Design and chemical synthesis of a sequence-specific DNA-cleaving protein

Abstract

We report the design and chemical synthesis of a sequence-specific DNA-cleaving protein consisting wholly of naturally occurring α-amino acids. The tripeptide, H-glyglyhis-OH (GGH), which is a consensus sequence for the copper-binding domain of serum albumin, was attached to the amino terminus of the DNA-binding domain of Hin recombinase (residues 139-190) to afford a new 55-residue protein, GGH(Hin 139-190) with two structural domains each with distinct functions, sequence-specific recognition, and cleavage of double helical DNA (Figure 1). The designed protein was synthesized by solid-phase techniques and shown, by footprinting, to be competent to bind at pM concentrations to four Hin sites, each 13 base pairs in length. In the presence of Cu(II), hydrogen peroxide, and sodium ascorbate, strong cleavage of DNA by GGH(Hin 139-190) occurs at one of the four sites by oxidative degradation of the deoxyribose backbone.

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