Recognition of specific DNA sequences by the c-myb protooncogene product: role of three repeat units in the DNA-binding domain.

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Abstract
The DNA-binding domain of c-Myb consists of three homologous tandem repeats of 52 amino acids. The structure of the third (C-terminal) repeat obtained by NMR analysis has a conformation related to the helix-turn-helix motif. To identify the role of each repeat in the sequence recognition of DNA, we analyzed specific interactions between c-Myb and DNA by measuring binding affinities for systematic mutants of Myb-binding DNA sites and various truncated c-Myb mutants. We found that specific interactions are localized unevenly in the AACTGAC region in the consensus 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 other hand, the interaction at the second adenine is less specific, with the affinity reduction in the range of 6- to 15-fold. The seventh cytosine involves a rather peculiar interaction, in which only guanine substitution abolishes the specific binding. The binding analyses, together with the chemical protection analyses, showed that the c-Myb fragment containing the second and third repeats covers the AACTGAC region from the major groove of DNA in such an orientation that the third repeat covers the core AAC sequence. These results suggest that the third repeat recognizes the core AAC sequence very specifically, whereas the second repeat recognizes the GAC sequence in a more redundant manner. The first (N-terminal) repeat, which covers the major groove of DNA only partially, is not significant in the sequence recognition, but it contributes to increase the stability of the Myb-DNA complex. The presence of an N-terminal acidic region upstream of the first repeat, which 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.