THE YEAST ACTIVATOR HAP1 - A GAL4 FAMILY MEMBER - BINDS DNA IN A DIRECTLY REPEATED ORIENTATION

The yeast transcriptional activator HAP1 contains a DNA-binding domain homologous to GAL4, PPR1, and related factors. By selecting random HA P1-binding sites, we found that HAP1, like GAL4, binds to two CGG trip lets. Unlike GAL4, the CGGs in the HAP1 consensus are in a direct and not inverted orientation. Sites with inverted CGGs were not recovered, and mutations converting the direct repeat of CGGs to an inverted rep eat greatly reduce HAP1-binding affinity. Also, the 6-bp spacer betwee n the CGGs contains a consensus TA that is positioned asymmetrically. Dimethylsulfate protection patterns on six of these sites show protect ions and enhancements that also lie in a directly repeated orientation , suggesting that the two HAP1 DNA recognition domains of a HAP1 homod imer are oriented in a directly repeated configuration on the DNA. Mor eover, substitution of the HAP1 dimerization domain with that of PPR1, which forms coiled-coils and dimerizes symmetrically, did not diminis h the ability of the protein to bind selectively to a direct repeat. T his result suggests that one DNA-binding domain of the HAP1 homodimer must be able to swivel 180 degrees relative to the dimerization domain to make specific contacts with the second CGG triplet. Our results pr esent a novel example of domain swiveling in one of the two identical subunits of a homodimer to accommodate specific DNA contacts to both C GG triplets of a direct repeat.