Principles and methods of affinity cleavage in studying transcription.
Explore the source record for details and available documents.
Binding Sites↗
Biomedical subjects
Brian D Schmidt
Publications and source records attributed to Brian D Schmidt.
Proteolytic DNA for mapping protein-DNA interactions.
We describe a technique to determine sites on proteins involved in protein-DNA interactions. DNA was synthesized via polymerase chain reaction (PCR) to produce four polynucleotide products with phosphorothioate nucleotides at the A, T, G, or C residues. Limited conjugation with the chemical protease FeBABE results in the surface of DNA being randomly labeled at the phosphorothioate sites with this protein-cleaving reagent. After formation of a protein-DNA complex, the proteolytic DNA can be activated to cleave the protein backbone at sites near the DNA. This technique was used to study the bacterial RNA polymerase/lacUV5 DNA open promoter complex, about which significant structural information is available. Cleavage sites on the two largest subunits of RNA polymerase, beta and beta', agree well with a recent model based on the crystal structure of the core enzyme alpha(2)betabeta' [Naryshkin, N., Revyakin, A., Kim, Y., Mekler, V., and Ebright, R. H. (2000) Cell 101, 601-611]. The cleavage site present on alpha supports previous studies regarding DNA binding regions of the alpha subunit. Cleavage sites identified throughout the sigma(70) subunit help to orient it with respect to the open promoter complex.
Bacterial Proteins↗
Electrophilic chelating agents for irreversible binding of metal chelates to engineered antibodies.
Radiolabeled monoclonal antibodies are widely used in the detection and treatment of cancer. However, several problems still prevent full clinical exploitation of these reagents. Low tumor/background ratios in radioimmunoscintigraphy and high background radioactivity in therapy are the foremost among these. The strategy of pretargeting which separates the tumor-targeting step from radiolocalization step may overcome these limitations. One pretargeting approach, based on the streptavidin-biotin system, has been demonstrated to successfully treat cancer in preclinical models (Proc. Natl. Acad. Sci. 97 (2000) 1802). In this report we describe the synthesis of several electrophilic chelates, designed for use in vivo. In this new pretargeting approach, we have used protein engineering to prepare an antibody that can bind selectively and irreversibly to certain of these metal chelates. This improves upon approaches based on the immunogenic protein streptavidin and the endogenous ligand biotin.
Animals↗