RECOGNITION OF SPECIFIC DNA-SEQUENCES BY THE C-MYB PROTOONCOGENE PRODUCT - ROLE OF 3 REPEAT UNITS IN THE DNA-BINDING DOMAIN

The DNA-binding domain of c-Myb consists of three homologous tandem re peats of 52 amino acids. The structure of the third (C-terminal) repea t obtained by NMR analysis has a conformation related to the helix-tur n-helix motif. To identify the role of each repeat in the sequence rec ognition of DNA, we analyzed specific interactions between c-Myb and D NA by measuring binding affinities for systematic mutants of Myb-bindi ng DNA sites and various truncated c-Myb mutants. We found that specif ic interactions are localized unevenly in the AACTGAC region in the co nsensus binding site of c-Myb: The first adenine, third cytosine, and fifth guanine are involved in very specific interactions, in which any base substitutions reduce the binding affinity by >500-fold. On the o ther hand, the interaction at the second adenine is less specific, wit h the affinity reduction in the range of 6- to 15-fold. The seventh cy tosine involves a rather peculiar interaction, in which only guanine s ubstitution abolishes the specific binding. The binding analyses, toge ther with the chemical protection analyses, showed that the c-Myb frag ment containing the second and third repeats covers the AACTGAC region from the major groove of DNA in such an orientation that the third re peat covers the core AAC sequence. These results suggest that the thir d repeat recognizes the core AAC sequence very specifically, whereas t he second repeat recognizes the GAC sequence in a more redundant manne r. The first (N-terminal) repeat, which covers the major groove of DNA only partially, is not significant in the sequence recognition, but i t contributes to increase the stability of the Myb-DNA complex. The pr esence of an N-terminal acidic region upstream of the first repeat, wh ich is important for the activation of c-myb protooncogene, was found to reduce the binding affinity by interfering with the first repeat in binding to DNA.