title: Synthesis of a hybrid protein containing the iron-binding ligand of bleomycin and the DNA-binding domain of Hin.
abstract: 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 NH2 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.
authors.affiliation: Arnold and Mabel Beckman Laboratories of Chemical Synthesis, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
authors.forename: M G
authors.initials: MG
authors.lastname: Oakley
authors.forename: K D
authors.initials: KD
authors.lastname: Turnbull
authors.forename: P B
authors.initials: PB
authors.lastname: Dervan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001761:Bleomycin / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D001761:Bleomycin; D004247:DNA; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase