Nuclease digestion of circular TRP1ARS1 chromatin reveals positioned nucleosomes separated by nuclease-sensitive regions.
TRP1ARS1 is a circular yeast DNA of 1453 base-pairs that contains the N-5'phosphoribosyl anthranilate isomerase (TRP1) gene and a sequence important for autonomous replication (ARS1). It exists extrachromosomally in 100 to 200 copies/cell and is presumably packed in nucleosomes. TRP1ARS1 has been partially purified as chromatin from lysed spheroplasts of yeast using gel filtration. A structural analysis of mapping micrococcal nuclease and DNAase I cutting sites with an accuracy of +/- 20 base-pairs is presented. Comparison of nuclease cleavage sites in chromatin and in purified DNA reveals that regions which are protected against nuclease attack are not distributed randomly. These regions are big enough to accommodate nucleosome cores. Three nucleosomes are positioned in the so-called ARS sequences, and are stable at low and high levels of digestion. The TRP1 gene region is covered by four nucleosomes, but they are neither randomly arranged nor precisely positioned. They are not stable and rearrange or disintegrate during digestion. The nucleosomal regions are separated by two segments of DNA (A, B), each about 180 base-pairs long, which are very sensitive to DNAase I and micrococcal nuclease and therefore presumably not packed in nucleosomes. Region B is found 5' to the TRP1 gene and might be related to transcription, whereas region A is centered around the termination codon of the TRP1 gene and the putative origin of replication.
D019747:Aldose-Ketose Isomerases; D001482:Base Composition; D002238:Carbohydrate Epimerases / Q000235:genetics; D002843:Chromatin / Q000302:isolation & purification; D002874:Chromosome Mapping; D004261:DNA Replication; D004270:DNA, Circular / Q000302:isolation & purification; D004271:DNA, Fungal / Q000302:isolation & purification; D003850:Deoxyribonuclease I; D004587:Electrophoresis, Agar Gel; D005796:Genes; D008836:Micrococcal Nuclease; D009707:Nucleosomes; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic
D002843:Chromatin; D004270:DNA, Circular; D004271:DNA, Fungal; D009707:Nucleosomes; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase