Synthesis of a hybrid protein containing the iron-binding ligand of bleomycin and the DNA-binding domain of Hin

The iron-binding and oxygen-activating domain of the natural product bleomycin [pyrimidoblamic acid-beta-hydroxy-L-histidine (PBA-beta-OH-His)] was attached to the NH_2 terminus of the DNA binding domain of Hin recombinase (residues 139-190). This hybrid 54-residue protein PBA-beta-OH-His-Hin-(139-190) binds specifically to DNA at four distinct Hin binding sites with affinities comparable to those of the unmodified Hin(139-190). In the presence of dithiothreitol, Fe(II).PBA-beta-OH-His-Hin-(139-190) cleaves DNA with specificity remarkably similar to that of Fe(II).EDTA-Hin(139-190). Analysis of the cleavage patterns suggests that site-specific DNA cleavage is mediated by a localized diffusible species, in contrast with cleavage by bleomycin, which occurs through a nondiffusible oxidant. This has implications for the design of second-generation artificial sequence specific DNA cleaving proteins and defines limitations in current efforts to create atom-specific chemistry on DNA