Sequence requirement for specific interaction of an enhancer binding protein (EBP1) with DNA

Short DNA sequence motifs have been identified in viral and cellular enhancers which represent the binding sites for a variety of trans- acting factors. One such HeLa cell factor, EBP1, has been purified and shown to bind to sequences in the SV40 enhancer. The PRDII element in the human beta-interferon gene regulatory element (IRE) shows strong sequence similarity to the EBP1 binding site in the SV40 enhancer. We demonstrate here that EBP1 binds to its sites in the SV40 enhancer and IRE in a similar manner, making base specific contacts over one complete turn of the DNA double helix. Mutational analysis of the EBP1 sites in the IRE and SV40 enhancer has identified the DNA sequence requirements necessary for specific EBP1/DNA complex formation. In addition, 34 DNA sequences related to the EBP1 binding site were analysed for their ability to bind EBP1. Sequences constituting high affinity binding sites possess the sequence 5'-GG(N)6CC-3'. Single base pair changes in the region between the conserved Gs and Cs can generally be tolerated although it is clear that these intervening bases contribute to binding affinity. Mutations in the recognition site which could lead to gross structural changes in the DNA abolish EBP1 binding.