The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle.

Abstract

The chromatin structures of two well-characterized autonomously replicating sequence (ARS) elements were examined at their chromosomal sites during the cell division cycle in Saccharomyces cerevisiae. The H4 ARS is located near one of the duplicate nonallelic histone H4 genes, while ARSI is present near the TRP1 gene. Cells blocked in G, either by a-factor arrest or by nitrogen starvation had two DNase I-hypersensitive sites of about equal intensity in the ARS element. This pattern of DNase I-hypersensitive sites was altered in synchronous cultures allowed to proceed into S phase. In addition to a general increase in DNase I sensitivity around the core consensus sequence, the DNase I-hypersensitive site closest to the core consensus became more nuclease sensitive than the distal site. This change in chromatin structure was restricted to the ARS region and depended on replication since cdc7 cells blocked near the time of replication initiation did not undergo the transition. Subsequent release of arrested cdc7 cells restored entry into S phase and was accompanied by the characteristic change in ARS chromatin structure.Autonomously replicating sequence (ARS) elements of the yeast Saccharomyces cerevisiae were first identified by their ability to enable plasmids to replicate as independent episomal minichromosomes (26,64,65). Considerable evidence has accumulated to suggest that ARS elements are origins of DNA replication (reviewed in references 42 and 71). For example, the replication of ARS plasmids take places only during S phase, is under the same genetic control as chromosomal replication, and occurs only once per division cycle (16,77,78). Electron microscopy of the 2 ,Lm plasmid (43) and the ribosomal DNA locus (50) showed that at least some of the mapped replication bubbles were coincident with known ARS elements. Recently, in vivo replication origins have been localized by two-dimensional gel mapping techniques to the 2,um plasmid ARS (6, 27), ARSI on a plasmid (6), the chromosomal ribosomal DNA ARS (35), an ARS on chromosome III called A6C (28), and ARSI on the chromosome (18).A detailed molecular examination of at least five different ARS elements has led to a consistent picture of the DNA sequence requirements for function (3,5,7,9,32,33,45,62). The ARS element is a bipartite structure composed of an 11-bp core consensus sequence and a 3' flanking domain of approximately 60 bp. Distal sequences are not required for replication activity, although they may influence efficiency depending on the particular ARS. The core consensus was first detected by comparative sequence analysis (7, 63), and its importance has since been rigorously demonstrated by point mutations, deletions, and linker-scanning mutations. The requirement for a 3' flanking domain has been established by deletion analysis; however, unlike the core consensus, the 3' flanking domain does not depend on an absolute primary DNA sequence (3,45 Chromatin structure may play an important role in the function of replication origins. Several origins h...

Saccharomyces telomeres assume a non-nucleosomal chromatin structure.

“…This result is consistent with the possibility that Y' junctions are also hypersensitive to DNase I. In yeast, DNase I hypersensitive sites are found flanking centromeres (Bloom and Carbon 1982), at ARSs (Thoma et al 1984;Brown et al 1991), and at other sites bound by nonhistone proteins such as at the GALIJO upstream activating sequence (UAS) (Lohr 1984). Thus, DNase I hypersensitivity at telomeres and at other genomic sites may mark transition points between different chromatin structures.…”

A Novel Histone H4 Mutant Defective in Nuclear Division and Mitotic Chromosome Transmission

“…Flow cytometry was carried out on cells fixed with ethanol and stained with propidium iodide as described previously (17,78). For G 1 arrest, cells were blocked by nitrogen starvation, as described previously (13), until more than 85% of cells were unbudded.…”

In vivo protein-DNA interactions at human DNA replication origin.

“…Replication origins in yeast (35,36) (20,38). Two DNA-nicking reagents were used, DNase I (which can be utilized only with isolated nuclei) and DMS (a small molecule that can be used with intact cells).…”