Transcription through the yeast origin of replication ARS1 ends at the ABFI binding site and affects extrachromosomal maintenance of minichromosomes

The Saccharomyces cerevisiae hyperrecombination mutation hpr1⌬ results in instability of sequences between direct repeats that is dependent on transcription of the repeat. Here it is shown that the HPR1 gene also functions in plasmid stability in the presence of destabilizing transcription elongation. In the hpr1⌬ mutant, plasmid instability results from unchecked transcription elongation, which can be suppressed by a strong transcription terminator. The plasmid system has been used to examine in vivo aspects of transcription in the absence of Hpr1p. Nuclear run-on studies suggest that there is an increased RNA polymerase II density in the hpr1⌬ mutant strain, but this is not accompanied by an increase in accumulation of cytoplasmic mRNA. Suppression of plasmid instability in hpr1⌬ can also be achieved by high-copy expression of the RNA splicing factor SUB2, which has recently been proposed to function in mRNA export, in addition to its role in pre-mRNA splicing. High-copy-number SUB2 expression is accompanied by an increase in message accumulation from the plasmid, suggesting that the mechanism of suppression by Sub2p involves the formation of mature mRNA. Models for the role of Hpr1p in mature mRNA formation and the cause of plasmid instability in the absence of the Hpr1 protein are discussed.

Section: Resultssupporting

confidence: 93%

Role of Transcription in Plasmid Maintenance in the hpr1Δ Mutant of Saccharomyces cerevisiae

Merker

Klein

2002

“…This is also where CUP1 was inserted and probably accounts for the less ordered structure of the ARS1 region in TAC. Another relevant factor is the much higher copy number of TRP1 ARS1 (100 to 200 per cell) (62) relative to TAC and other TRP1 ARS1 plasmids with inserts (20,53): most TRP1 genes in TRP1 ARS1-containing (Fig. 6).…”

Section: Discussionmentioning

confidence: 99%

Remodeling of YeastCUP1Chromatin Involves Activator-Dependent Repositioning of Nucleosomes over the Entire Gene and Flanking Sequences

Shen

LeBlanc

Alfieri

et al. 2001

The yeast CUP1 gene is activated by the copper-dependent binding of the transcriptional activator, Ace1p. An episome containing transcriptionally active or inactive CUP1 was purified in its native chromatin structure from yeast cells. The amount of RNA polymerase II on CUP1 in the purified episomes correlated with its transcriptional activity in vivo. Chromatin structures were examined by using the monomer extension technique to map translational positions of nucleosomes. The chromatin structure of an episome containing inactive CUP1 isolated from ace1⌬ cells is organized into clusters of overlapping nucleosome positions separated by linkers. Novel nucleosome positions that include the linkers are occupied in the presence of Ace1p. Repositioning was observed over the entire CUP1 gene and its flanking regions, possibly over the entire episome. Mutation of the TATA boxes to prevent transcription did not prevent repositioning, implicating a chromatin remodeling activity recruited by Ace1p. These observations provide direct evidence in vivo for the nucleosome sliding mechanism proposed for remodeling complexes in vitro and indicate that remodeling is not restricted to the promoter but occurs over a chromatin domain including CUP1 and its flanking sequences.

“…Alternatively, strong activators could induce abortive transcription from neighboring cryptic promoters, resulting in inhibition of replication. Indeed, transcription entering ARS1 inhibits DNA replication (42,47). The presence of a selectable marker gene with strong promoter activity close to the ARS might counteract the recruitment of transcription machinery to the ARS itself, thereby relieving the negative effect of certain transcription factors on DNA replication.…”

Section: Discussionmentioning

confidence: 99%

Context-Dependent Modulation of Replication Activity ofSaccharomyces cerevisiaeAutonomously Replicating Sequences by Transcription Factors

Kohzaki

Ito

Murakami

1999

Evidence for transcription factor involvement in the initiation of DNA replication at certain replication origins in Saccharomyces cerevisiae mainly comes from an indirect assay which measures the mitotic stability of plasmids containing an autonomously replicating sequence (ARS), a selectable marker gene, and a centromere. In order to eliminate the effect of transcription factor binding to the selectable marker gene or centromere in such assays, we have adapted the DpnI assay to directly measure ARS replication activity in vivo by using ARS plasmids devoid of extraneous transcription elements. Using this assay, we found that the B3 element of ARS1, which serves as a binding site for the transcription factor Abf1p, does not stimulate ARS activity on plasmids lacking a centromere and a selectable marker gene. We also found with such plasmids that exogenous expression of the strong transcriptional activators Gal4 and Gal4-VP16 inhibited the replication activity of ARS1 when B3 was replaced by the Gal4 binding site, although these activators had previously been shown to stimulate replication activity in the stability assay. Moreover, a chromosomally inactive ARS, ARS301, which was active by itself on a plasmid, was inactivated by placing an Abf1p binding site in its vicinity. These results indicate that the sequences surrounding the ARS as well as properties of the ARS element itself determine its response to transcription factors.Saccharomyces cerevisiae provides an excellent system for the analysis of eukaryotic replication origins for several reasons. First of all, the replication origin can be identified on a functional basis by analyzing it for DNA sequences that replicate in an autonomous fashion as plasmid DNA in yeast cells (autonomous replicating sequence [ARS]). When a DNA fragment with origin activity is ligated to a selectable marker gene, the resulting plasmid can transform yeast cells with high frequency (19,43). Secondly, relative replication activity is easily estimated by measuring the mitotic stability of each ARS plasmid (24). This method has revealed that replication origins of budding yeast are compact (100 to 200 bp), with a modular structure consisting of an essential "core" sequence and auxiliary sequences which modulate replication activity. For example, ARS1, one of the best-characterized origins, contains two elements, A and B (10, 30). The A element contains a small sequence that is essential for origin activity and conserved among all ARS sequences (ARS consensus sequence [ACS]) (32). The A element functions as a recognition site for the origin recognition complex (ORC) involved in the initiation of DNA replication (3, 4). The B element consists of three subelements, B1, B2, and B3, each of which contributes to efficient replication although none is essential (30). One function of the B1 element is to enhance the binding of the ORC to the A element (34-36). The function of B2 remains unclear. The B3 element is a binding site for the transcription factor Abf1p (30). Other ARS elements so...