title: Effects of the A.T/T.A degeneracy of pyrrole--imidazole polyamide recognition in the minor groove of DNA.
abstract: Pairing rules have been developed to predict the sequence specificity of minor groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids. An Im/Py pair distinguishes G.C from C.G and both of these from A.T/T.A base pairs. A Py/Py pair appears not to distinguish A.T from T.A base pairs. To test the extent of this degeneracy, the affinity and binding orientation of the hairpin polyamide ImPyPy-gamma-PyPyPy-beta-Dp were measured for eight possible five base pair 5'-TG(A,T)(3)-3' match sites. Affinity cleavage experiments using a polyamide with an EDTA.Fe(II) moiety at the carboxy terminus, ImPyPy-gamma-PyPyPy-beta-Dp-EDTA.Fe(II), are consistent with formation of an oriented 1:1 hairpin polyamide complex at all eight 5'-TG(A,T)(3)-3' binding sites [20 mM HEPES, 200 mM NaCl, 50 mg/ml glycogen, pH 7.0, 22 degrees C, 5 mM DTT, 1 mM Fe(II)]. Quantitative DNase I footprint titration experiments reveal that ImPyPy-gamma-PyPyPy-beta-Dp binds all eight 5'-TG(A,T)(3)-3' target sites with only a 12-fold difference in the equilibrium association constants between the strongest site, 5'-TGTTT-3' (Ka = 2.1 x 10(8) M-1), and the weakest site, 5'-TGAAT-3' (Ka = 1.8 x 10(7) M-1) (10 mM Tris.HCl, 10 mM KCl, 10 mM MgCl2, 5 mM CaCl2, pH 7.0, 22 degrees C). This relatively small range indicates that the Py/Py pair is approximately degenerate for recognition of A,T base pairs, affording generality with regard to targeting sequences of mixed A.T/T.A composition.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
authors.forename: S
authors.initials: S
authors.lastname: White
authors.forename: E E
authors.initials: EE
authors.lastname: Baird
authors.forename: P B
authors.initials: PB
authors.lastname: Dervan
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D004492:Edetic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007093:Imidazoles / Q000378:metabolism; D058085:Iron Compounds / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009757:Nylons / Q000737:chemistry / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism
chemical_list: D007093:Imidazoles; D058085:Iron Compounds; D009757:Nylons; D009838:Oligodeoxyribonucleotides; D011758:Pyrroles; D004247:DNA; D004492:Edetic Acid; D003850:Deoxyribonuclease I