Studies on DNA-binding peptides

Umurtak, Hatice Bahar (1989) Studies on DNA-binding peptides. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The specific recognition of DNA sequences by EcoR I, the cro and λ repressor proteins, trp repressor and zinc fingers is reviewed. The key structural features which mediate DNA-binding are discussed. Based on these observations, the design of a model system is described. Three requirements for experimentation are the availability of synthetic peptides, the preparation of a suitable DNA fragment and methods for detecting DNA-peptide interaction. Solid phase peptide synthesis and its application to the preparation of a series of peptides (12), (32), (33), (34), whose sequences were taken from the putative helical binding region of the cro and λ repressor proteins is outlined. Subsequent isolation and purification of a DNA substrate containing the natural O_R1 operator sequence is then discussed. Current radiochemical methods for assaying the binding specificity of small molecules to DNA are reviewed with particular emphasis upon DNA-footprinting and affinity cleavage. Initial binding studies revealed that, in aqueous buffer the peptides bound to the DNA indicating non-specific recognition. Subsequent work in which organic solvents were used to control peptide conformation is outlined, and CD evidence for secondary structural preferences in the test peptides is presented. Although in such solvent systems, the peptides did interact with double-stranded DNA, no unambigous evidence for sequence-specific binding could be obtained. Possible reasons for the observed behaviour are discussed.

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