NONO ENHANCES THE ASSOCIATION OF MANY DNA-BINDING PROTEINS TO THEIR TARGETS

NonO is an unusual nucleic acid binding protein not only in that it bi nds both DNA and RNA but that it does so via functionally separable do mains. Here we document that NonO enhances the binding of some (E47, O TF-1 and OTF-2) but not all (PEA3) conventional sequence-specific tran scription factors to their recognition sites in artificial substrates as well as in an immunoglobulin V-H promoter. We also show that NonO i nduces the binding of the Ku complex to DNA ends. Ku has no known DNA sequence specificity. These enhancement of binding effects are NonO co ncentration dependent, Using the E box activity of E47 as a model, kin etic studies demonstrate that the association rate of the protein-DNA complex increases in the presence of NonO while the dissociation rate remains the same, thereby increasing the sum total of the interaction. Oligo competition experiments indicate that NonO does not contact the target DNA in order to enhance the binding activity of DNA binding pr oteins. Rather, methylation interference analysis reveals that the ind uced E47 binding-activity has the same DNA-binding sequence specificit y as the normal binding. This result suggests that one of the effects of NonO is to induce a true protein-DNA interaction, In this way, it m ight be possible for NonO to play a crucial role in gene regulation.