Reevaluation of a DNA binding activity with specificity for chemically modified DNA

Abstract

A DNA binding activity which appeared in direct filter binding assays to show specificity for DNA modified by N-acetoxy-2-acetylaminofluorene (AAF), N-methyl-N-nitrosourea and methylmethanesulfonate (Moranelli and Lieberman, Proc. Natl. Acad. Sci. USA 77, 3201, 1980) has been further purified and characterized. Equilibrium competition binding experiments indicate this activity shows little, or no, preferential binding of AAF-modified DNA compared to single-stranded DNA and only a small preference compared to u.v.-irradiated DNA or double-stranded DNA. These findings demonstrate the difficulty of using direct filter binding assays when purifying proteins with a desired biological function and the need to perform competition assays when evaluating specificity.

Properties and purification of a colony‐stimulating factor of granulocytes and macrophages produced by mouse spleen cells

“…Each fraction was dialyzed against TMP buffer (10 mM Tris-HC1, pH 7.5; 1 mM MgClZ; 0.01% polyethylene glycol, MW 6,000) overnight at 4°C before loading onto a 1.5 cm x 10 cm column packed with DEAE-Bio Gel A (BioRad, Richmond, CA) and pre-equilibrated with TMP buffer. Chromatography was carried out according to the method described previously (Chou et al, 1982). The void volume was measured by monitoring the absorbance of the eluate at 276nm wavelength (O.D.…”