Positive DNA supercoiling generates a chromatin conformation characteristic of highly active genes

Abstract

During transcription, positive DNA supercoils generated ahead of RNA polymerase could theoretically uncoil the negative DNA supercoils associated with nucleosomes and thereby decondense the chromatin fiber in preparation for RNA polymerase passage. Here we examine the effect of positive DNA supercoiling on the structure of yeast 2-microns minichromosomes. We utilized a conditional topoisomerase mutant expressing Escherichia coli topoisomerase I to convert the DNA supercoiling state from negative to positive in vivo. Minichromosomes containing positively supercoiled DNA exhibited a striking increase in DNase I sensitivity. They also displayed additional micrococcal nuclease cleavage sites but yielded nearly typical nucleosomal ladders after extensive digestion. Upon in vitro relaxation with eukaryotic topoisomerase I, the minichromosomes remained DNase I sensitive but were converted to negative DNA supercoiling with a slightly increased linking number compared to typical minichromosomes, thus indicating the presence of bound histones. Therefore, positive DNA supercoiling provides a mechanism for generating, but is not required for maintaining, a conformation in chromatin characteristic of highly transcribed genes.