A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide.
An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.
Herbert
A G
AG
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Rich
A
A
D000818:Animals; D001973:Bromodeoxyuridine; D003432:Cross-Linking Reagents; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003849:Deoxyguanosine; D004591:Electrophoresis, Polyacrylamide Gel; D006041:Goats / Q000276:immunology; D015151:Immunoblotting; D007202:Indicators and Reagents; D015636:Magnesium Chloride; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D013698:Templates, Genetic; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D015636:Magnesium Chloride; D004247:DNA; D001973:Bromodeoxyuridine; D003849:Deoxyguanosine