Analysis of the structure of a natural alternating d(TA)n sequence in yeast chromatin.

Abstract

We address here the question of the in vivo structure of a natural alternating d(TA)n sequence found at the 3' region of the Saccharomyces cerevisiae FBP1 gene. This sequence consists of 13 TA pairs interrupted by a TT dinucleotide in the middle of the tract. Previous experiments with cruciform-specific nucleases S1 and Endonuclease VII demonstrated the presence in vitro of a cruciform in this region. We also showed this region to be part of a nuclease hypersensitive site flanked by nucleosomes in yeast chromatin. Here we demonstrate, by means of S1 in vivo footprinting, that in yeast plasmids also adopts in vivo a non B-DNA structure where is not a cruciform. A theoretical analysis of this region that it contains a site susceptible to superhelical stress duplex destabilization. The locations and conditions under which alternative structures form in the wild-type sequence and in deletion mutants agree with these theoretical predictions, suggesting that some kind of denaturation is the alternative structure adopted by the sequence in vivo. This suggests that negative superhelical stress sufficient for local denaturation exists in nucleosomal DNA. We also demonstrate by micrococcal nuclease digestions that the deletion of the alternating d(TA)n sequence modifies the chromatin hypersensitive site but does not affect nucleosome positioning.