IDENTIFICATION OF INDISPENSABLE RESIDUES FOR SPECIFIC DNA-BINDING IN THE IMPERFECT TANDEM REPEATS OF C-MYB R2R3

The individual repeats, R2 and R3, of the minimum specific DNA-binding domain (R2R3) of c-Myb have very similar structures, with a helix-tur n-helix variation motif, although their sequence identity in the tande m repeats is only 31%. From previous mutational and structural studies , the third helices in both repeats were shown to directly recognize t he specific base sequence, PyAAC(G)/(T)G. In order to elucidate the re ason for the imperfection of the tandem repeats at amino acid position s other than the recognition helices, a series of R2R3 mutants was gen erated by swapping the helices and the N-terminus in R2 to those in R3 . Consequently, the sequence composing the first helix of R2 was found to be essential for specific DNA-binding, in addition to the third re cognition helix of R2. Further mutational studies revealed that the on ly indispensable residues in the first helix are Val103 and Val107, wh ich are involved in the hydrophobic core of R2. These residues do not directly interact with the DNA, but they contribute to the correct for mation of helix 1 and the characteristic packing of R2, which is sligh tly different from that of R3, and are required for specific base reco gnition through strong cooperativity with R3.