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Nucleic Acids Research, 1988, Vol. 16, No. 17 8277-8289
© 1988

Articles

Detection of Z DNA binding proteins in tissue culture cells

I.R. Leith, R.T. Hay and W.C. Russell

Department of Biochemistry and Microbiology, University of St Andrews Irvine Building, North Street, St Andrews, Fife KY16 9AL, UK

Received July 4, 1988. Accepted August 9, 1988.

A gel electrophoresis DNA binding assay to detect Z DNA binding proteins has been developed utilising [32P] labelled poly [d(G-C)] which was converted to the Z form by incubation in 100 µM Co(NH3)6C13. The parameters of the assay were established using a Z DNA antibody as a model system and then applied to extracts of Hela and BHK21 cells. Using an anti-Z DNA antibody conditions were established which allowed resolution of antibody-DNAcomplexes and free DNA in the presence of 100 µM Co(NH3)6C13. The inclusion of unlabelled complementary homopolymers eliminated non-specific binding to the labelled Z-DNA probe. Competition experiments demonstrated that the assay was highly specific for double stranded non-B DNA. Application of the technique to extracts of mammalian cells demonstrated that human and hamster cells contain Z-DNA binding proteins; further characterisation by a blotting technique indicated that a 56,000 molecular weight cell protein preferentially binds Z-DNA.


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