Isolation and characterization of Z-DNA binding proteins from wheat germ.
The preparation of a heterogeneous non-histone protein extract from wheat germ utilizing Br-poly(dG-dC).poly(dG-dC) (Z-DNA) affinity chromatography is described. The binding characteristics of antibodies against Z-DNA are used as a model system to define important criteria that the DNA binding behavior of a Z-DNA binding protein should display. We show that the wheat germ extract contains DNA binding proteins specific for left-handed Z-DNA by these criteria. The affinity of the proteins measured by competition experiments was approximately 10(5) greater for Br-poly(dG-dC).poly(dG-dC) (Z-DNA) than for poly(dG-dC).poly(dG-dC) (B-DNA). The affinity of the proteins for plasmid DNA increases with increasing negative superhelicity which is known to stabilize Z-DNA. The proteins are shown to compete with Z-DNA antibodies for binding to supercoiled plasmids. Finally, the affinity for two plasmids at a given superhelical density is greater for the plasmid containing an insert known to form Z-DNA than for a plasmid without the insert. The proteins exhibit a 2-3-fold greater affinity for stretches of (dC-dA)n.(dT-dG)n over stretches of (dG-dC)n.(dG-dC)n when both sequences are induced to form Z-DNA by supercoiling.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D011863:Radioimmunoassay; D012639:Seeds / Q000378:metabolism*; D013950:Thymus Gland; D014908:Triticum / Q000378:metabolism
D004268:DNA-Binding Proteins; D011089:Polydeoxyribonucleotides; C019530:poly(dC-dG); D004247:DNA