Isolation of a condensed, intracellular form of the 2-micrometer DNA plasmid of Saccharomyces cerevisiae.

Livingston, Dennis; Hahne, Solveig

doi:10.1073/pnas.76.8.3727

Cited by 71 publications

(24 citation statements)

References 28 publications

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“…Thus, the aberrant chromatin structure of the A ORF is not an inherent property of the plasmid, but instead reflects the absence of 2,um-encoded factors. In contrast to (21,29,31). More recent studies indicate a nonrandom distribution of chromatin proteins; the direct repeats ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

Veit¹,

Fangman²

1985

Mol. Cell. Biol.

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We have used gene disruptions and nuclease probes to assess the roles of yeast 2,Im plasmid genes in plasmid chromatin organization. The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D. While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin. Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C. Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) controls copy number by regulating the level of the gene A recombinase.The 2,um plasmid is a 6,318-base-pair circular DNA found in most strains of Saccharomyces cerevisiae at 50 to 100 copies per diploid cell (for reviews, see references 4 and 5). Composing up to 2% of a relatively small nuclear genome, the plasmid offers a useful system for physical studies of eucaryotic DNA replication. Although multiple copy and episomal, the replication of the 2,um plasmid is like that of chromosomal DNA, with replication depending on the same set of chromosomally encoded genes (22) and each plasmid molecule replicating once during the S phase (37).Factors encoded by the 2,um plasmid itself are required for its stable maintenance. The plasmid contains several large open reading frames (ORFs), the five largest designated A, B, C, D, and E (16; see Fig. 1). Since the 2,um plasmid confers no easily scored phenotype on its host, investigations of the relationship between plasmid-encoded factors and plasmid maintenance have relied on 2,um derivatives that contain selectable nuclear genes. Stability studies of such chimeras, in which specific 2,um sequences are deleted or disrupted, indicate that the B and C ORFs (also referred to as REP1 and REP2, respectively) encode trans-acting factors necessary for stable plasmid maintenance (17,19). STB and ORI, two cis-acting sites defined by deletion analysis, are also essential for plasmid stability. STB, bounded by the PstI and AvaI sites (19), appears to be equivalent to the REP3 locus described by Jayaram et al. (17). ORI is defined by its ability to support the autonomous replication of plasmids (7,17). Electron microscopy studies of 2Rm plasmid replicative intermediates show a high frequency of eye forms centered about ORI, supporting the notion that ORI acts as an origin of replication (30).It is less clear whether other 2,um plasmid components are involved in plasmid maintenance. Although 2,um chimeric plasmids containing large insertions in the A or D ORFs are lost 10 to 100 times faster than is native 2,um plasmid (14, 15), it is unclear that the instability reflects the disruption of a component normally involved in plasmid stabilization. Unlike the instability resulting from the disruption of the B or C ORFs, ins...

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Section: Resultsmentioning

confidence: 99%

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

Veit¹,

Fangman²

1985

Mol. Cell. Biol.

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“…This demonstrates that plasmid segregation rather than replication is primarily affected in rsc2 mutants. 2m plasmid is condensed into 21 to 34 nucleosomes per plasmid that closely resemble those of chromosomal DNA (26). The protection of 2m chromatin from nuclease digestion is not random within the plasmid (25), and in particular the STB region has an abnormal structure (15,50).…”

Section: Discussionmentioning

confidence: 99%

RSC2, Encoding a Component of the RSC Nucleosome Remodeling Complex, Is Essential for 2μm Plasmid Maintenance in Saccharomyces cerevisiae

Wong

Scott-Drew

Hayes

et al. 2002

Molecular and Cellular Biology

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The stable maintenance of the 2m circle plasmid depends on its ability to overcome intrinsic maternal inheritance bias, which in yeast normally results in the failure to transmit DNA molecules efficiently to daughter cells. In addition to the plasmid proteins Rep1 and Rep2 acting on the plasmid DNA locus STB, it is likely that other chromosomally encoded yeast proteins are required. We have isolated mutants of yeast unable to maintain 2m and found that RSC2 is essential for 2m to overcome maternal inheritance bias. Rsc2 is part of a multisubunit RSC chromatin remodeling complex, and we show that in the absence of Rsc2 the chromatin structure of the STB region is significantly altered and the Rep1 protein loses its normal localization to subnuclear foci. Rsc1, a closely related homolog of Rsc2 present in an alternative form of the RSC complex, is not required for 2m maintenance and does not replace the requirement for Rsc2 when overexpressed. This represents the first specific role for Rsc2 that has been related to a change in chromatin structure, as well as the first direct evidence linking chromatin structure to 2m segregation.The 2m circle is a high-copy-number plasmid widespread in laboratory and industrial strains of Saccharomyces cerevisiae that has been used as a molecular model for aspects of cellular function such as DNA replication, gene transcription, and recombination, as well as the basis of yeast cloning vectors. High-copy-number plasmids carrying only replication origins show maternal inheritance bias (MIB) and are poorly segregated to daughter cells (31), whereas 2m and its derivatives are effectively segregated between mother and daughter cells at division (22). However, the mechanism by which 2m escapes MIB resulting in equal segregation is not fully understood, nor have all of the host components involved been identified. Of the four genes carried by the plasmid, REP1 and REP2 are necessary for MIB to be overcome, and their products act at the plasmid locus STB to confer plasmid stability (2,10,18,22,33,36). Within the nucleus, Rep1 and Rep2 proteins are localized into discrete foci (39) in which the plasmid is also colocalized (39, 52). These plasmid and protein foci are segregated in a manner similar to that of the chromosomes themselves, a finding consistent with the observation that the 2m plasmid is packaged into nucleosomes (50). Thus, the plasmid may be exploiting the chromosomal segregation machinery indirectly by associating with specific or nonspecific chromosomal sites. It has been therefore suggested that the segregation of 2m, and hence its overcoming of MIB, likely requires host (non-plasmid-encoded) genes (18). One candidate is the product of the SHF1 (CST6) gene, which has been shown to bind to STB in vivo and in vitro (51).Nucleosomes regulate and modify the ability of proteins to interact with DNA, and the remodeling of chromatin and repositioning of nucleosomes is important in gene regulation and other functions of DNA. There is evidence that specific chromatin structures are...

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“…8A, left panel). In contrast, 2-|jLm DNA , which is nucleosomal (Livingston and Hahne 1979;Nelson and Fangman 1979), was not precipitated by the antiserum (Fig. 8A, right panel).…”

Section: Solubilized Telosomes Retain Bound Proteinmentioning

confidence: 98%

Saccharomyces telomeres assume a non-nucleosomal chromatin structure.

Wright¹,

Gottschling²,

Zakian³

1992

Genes Dev.

260

214

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The chromatin structures of the telomeric and subtelomeric regions on chromosomal DNA molecules in Saccharomyces cerevisiae were analyzed using micrococcal nuclease and DNase I. The subtelomeric repeats X and Y' were assembled in nucleosomes. However, the terminal tracts of Cj.jA repeats were protein protected in a particle larger than a nucleosome herein called a telosome. The proximal boundary of the telosome was a DNase I hypersensitive site. This boundary between the telosome and adjacent nucleosomes was completely accessible to Escherichia coli dam methylase when this enzyme was expressed in yeast, whereas a site 250 bp internal to the telomeric repeats was relatively inaccessible. Telosomes could be cleaved from chromosome ends with nuclease and solubilized as protein-DNA complexes. Immunoprecipitation of chromosomal telosomes with antiserum to the RAPl protein indicated that RAPl was one component of isolated telosomes. Thus, the termini of chromosomal DNA molecules in yeast are assembled in a non-nucleosomal structure encompassing the entire terminal C1.3A tract. This structure is separated from adjacent nucleosomes by a region of DNA that is highly accessible to enzymes.

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Isolation of a condensed, intracellular form of the 2-micrometer DNA plasmid of Saccharomyces cerevisiae.

Cited by 71 publications

References 28 publications

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

RSC2, Encoding a Component of the RSC Nucleosome Remodeling Complex, Is Essential for 2μm Plasmid Maintenance in Saccharomyces cerevisiae

Saccharomyces telomeres assume a non-nucleosomal chromatin structure.

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