Detection of Z DNA binding proteins in tissue culture cells.
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 microM Co(NH3)6Cl3. 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-DNA complexes and free DNA in the presence of 100 microM Co(NH3)6Cl3. 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.
Leith
I R
IR
Department of Biochemistry and Microbiology, University of St Andrews, Fife, UK.
Hay
R T
RT
Russell
W C
WC
D000906:Antibodies / Q000032:analysis; D002482:Cellulose / Q000031:analogs & derivatives; D004247:DNA / Q000031:analogs & derivatives / Q000276:immunology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides / Q000378:metabolism*
D000906:Antibodies; D004268:DNA-Binding Proteins; C033190:DNA-cellulose; D011089:Polydeoxyribonucleotides; C019530:poly(dC-dG); D002482:Cellulose; D004247:DNA