Evidence of an overlap between the two half-sites of UAS1-B/CYC1--a new model for Cyp1p (Hap1p) DNA binding.

Abstract

Cyp1p (Hap1p) is a yeast transcriptional regulator belonging to the zinc-cluster family. CGGNNNTANCGG was identified by PCR selection as the DNA sequence allowing its optimal binding. Nevertheless, this sequence is not a consensus sequence, the simultaneous presence of the two CGGs and the TA generally not being found in the known natural Cyp1p targets. In fact, our previous studies showed that the mechanism of Cyp1p DNA binding was target dependent. Data concerning the binding of Cyp1p to the UAS1-B/CYC1 are presented here. This target, containing the CGGGGTTTACGG sequence, was found to present the particular ability of stabilizing the binding of only one molecule of some monomeric Cyp1p fragments. This property was used to investigate the actual contribution of the TT and CGG sequences in the binding of Cyp1p. Our results indicate that each CGG belongs to a different half-site and, in contrast to a previous hypothesis, that the T nucleotide located four bases downstream from the left CGG is essential for the binding of one monomer to each half-site. The two half-sites of the UAS1-B/CYC1 thus overlap.