HOMEODOMAIN PROTEINS - WHAT GOVERNS THEIR ABILITY TO RECOGNIZE SPECIFIC DNA-SEQUENCES

Deformed (Dfd) and Ultrabithorax (Ubx) are homeodomain proteins from D rosophila melanogaster eu that exert regulatory effects on gene expres sion by binding to specific target sites in the fly genome using a hel ix-turn-helix (HTH) motif. The recognition helices of these two protei ns are almost identical and the DNA sequences they recognize are simil ar, containing a conserved TAAT core sequence flanked by a somewhat va riable sequence. Yet the in vivo functions of the two proteins are qui te different. We have used the homeodomains of these two proteins and in vitro selected DNA binding sites to characterize the structural det ails of homeodomain binding to DNA and to understand the basis for the differences in sequence specificity between homeodomains with similar recognition helices. We have employed hydroxyl radical cleavage of DN A to study the positioning of the proteins on the binding sites and ha ve analyzed the effects of missing nucleosides and purine methylation on homeodomain binding. Our results indicate that the positioning of t he Ubx and Dfd homeodomains on their binding sites is consistent with reported structures of other homeodomain/DNA complexes. Dfd and Ubx bi nd to DNA with the recognition helix in the major groove 3' to the TAA T core sequence and the N-terminal arm in the adjacent minor groove. H owever, we observe striking differences between the two homeodomains i n their specific interactions with DNA. Missing nucleosides within the selected binding sites have differential effects on protein binding, which are dependent on the identity of the homeodomain. Differences at the 3' end of the binding site on the top strand indicate that the N- terminal arm of a homeodomain is capable of distinguishing an A . T ba se-pair from T . A in the minor groove. Specific orientation of the N- terminal arm within the binding site appears to vary between the homeo domains and influences the interaction of the recognition helix with t he major groove. (C) 1995 Academic Press Limited