Direct simulation of yeast 2-μm circle plasmid amplification

Russo, Frank D.; Scherson, Isaac D.; Broach, James R.

doi:10.1016/s0022-5193(05)80604-6

Cited by 6 publications

(6 citation statements)

References 16 publications

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“…Even though formation of pince-nez chromosomes had been already proposed (Russo et al 1992;Smith 2012), and even reported in plasmids (Petes and Williamson 1994), possible mechanisms of their resolution are currently unknown. Thus, until such resolution mechanisms are found, tying up the replicating chromosome mass in pince-nez structures may be considered a lethal event and a general explanation for the toxicity of maximized CRC in recombinational repair-proficient cells.…”

Section: Recombinational Misrepairmentioning

confidence: 99%

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

Khan¹,

Mahaseth²,

Kouzminova³

et al. 2016

Genetics

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We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the "natural" CRC limit of 8 (cells divide more slowly); the "functional" CRC limit of 22 (cells divide extremely slowly); and the "tolerance" CRC limit of 64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter. KEYWORDS overinitiation; hydroxyurea; seqA; rep; recA E UKARYOTIC and prokaryotic chromosomes differ in many important aspects (Kuzminov 2014), and one key difference lies in the spatio-temporal organization of chromosomal replication. In contrast to eukaryotes, which perform multibubble replication (Masai et al. 2010), most bacteria replicate their singular chromosome in the unibubble format by initiating bidirectional replication from a designated replication origin (oriC) (Sernova and Gelfand 2008;Leonard and Méchali 2013) and finishing replication within a broad termination zone (ter) (Mirkin and Mirkin 2007;Duggin et al. 2008). Eukaryotes always perform a single replication round in their chromosomes (Masai et al. 2010;Diffley 2011), keeping the ratio of maximally replicated to unreplicated DNA in the same chromosome (the "replication complexity index") strictly at 2 ( Figure 1A). Due to the defined origin and terminus of prokaryotic chromosomes, chromosomal replication complexity in bacteria can be simply expressed as the ori/ter ratio ( Figure 1B). Even though unique cell cycles in some bacteria, such as Caulobacter, also maintain a strict CRC = 2 (Collier 2012), bacterial cells are generally recognized for their ability to support multiple concurrent replication rounds within the same chromosome (Morigen et al. 2009).In reality, under typical growth conditions the ori/ter ratio in exponentially growing bacterial cells is still 2 (Bird et al. 1972;Bipatnath et al. 1998;Wang et al. 2007;Murray and Errington 2008;Rotman et al. 2009;Stokke et al. 2011), showing that bacterial cells, like eukaryotes, also prefer to deal with a single replication round in their chromosomes. However, due to the peculiarity of the prokaryotic chromosome cycle (Kuzminov 2013), whe...

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Section: Recombinational Misrepairmentioning

confidence: 99%

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

Khan¹,

Mahaseth²,

Kouzminova³

et al. 2016

Genetics

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“…Plasmid amplification is absolutely dependent on the 2m circle Flp site-specific recombination system (33). A currently favored model for amplification proposes the recombinational inversion of a bidirectional replication fork and the resultant double-rolling-circle replication mode as the means for obtaining multiple replicas of the plasmid from a single initiation event (9)(10)(11)26). The cessation of amplification would require a second recombination event that can restore the normal direction of fork movement.…”

mentioning

confidence: 99%

The 2μm Plasmid Stability System: Analyses of the Interactions among Plasmid- and Host-Encoded Components

Velmurugan

Ahn

Yang

et al. 1998

Molecular and Cellular Biology

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The stable inheritance of the 2m plasmid in a growing population of Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In this study we demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely diminish their normal capacity to localize to the yeast nucleus. The nuclear targeting, as well as their functional role in plasmid partitioning, can be restored by the addition of a nuclear localization sequence to the amino or the carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstrate that the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vivo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting with the STB locus. The Shf1 protein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 locus, a 2m circle-derived plasmid shows relatively poor stability.The 2m circle, a relatively small circular plasmid (6,318 bp) present in most common strains of Saccharomyces cerevisiae, has optimized a partitioning system and an amplification system that allow it to be propagated stably in a cell population at a copy number of approximately 60 to 100 per cell (reviewed in reference 2). Genetic analyses suggest that two plasmid-coded proteins, Rep1p and Rep2p, in conjunction with a cis-acting locus STB (also called REP3) contribute to the stability function (16,17,19,23). One plausible mechanism for plasmid stability is that the interaction of Rep1p and Rep2p with the STB element serves to overcome the normal bias in plasmid segregation that tends to favor the mother cell over the daughter cell (22). The evidence for this suspected DNA-protein interaction is quite preliminary and rests almost entirely on the observation that urea-solubilized yeast extracts expressing Rep1p and Rep2p or [cir 0 ] extracts supplemented exogenously with Rep1p and Rep2p can bind STB (14).The need for plasmid amplification arises only if and when there is a decrease in copy number below the steady-state value. Normally, each plasmid molecule is replicated once, and only once, per cell cycle (35), and the daughter molecules are partitioned efficiently at cytokinesis (27). When there is a drop in copy number, the amplification system overrides the cell cycle restriction of a single round of plasmid replication during one S phase. Plasmid amplification is absolutely dependent on the 2m circle Flp site-specific recombination system (33). A currently favored model for amplification proposes the recombinational inversion of a bidirectional replication fork and the resultant double-rolling-circle replication mode as the means for obtaining multiple replicas of the plasmid fro...

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“…This amplification mechanism overrides the cell cycle restriction on replication and is dependent on the 2m circle F1p site-specific recombination system. A model for how the recombinational inversion of a bidirectional replication fork can result in multiple rounds of replication from a single initiation event has been proposed (6,7,23). Although the act of recombination has been shown to be indispensable in amplification (28), not all of the predictions of the Futcher model have been satisfactorily verified.…”

mentioning

confidence: 99%

The 2microm-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus

Ahn

Biswal

et al. 1997

J Bacteriol

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The efficient partitioning of the 2m plasmid of Saccharomyces cerevisiae at cell division requires two plasmid-encoded proteins (Rep1p and Rep2p) and a cis-acting locus, REP3 (STB). By using protein hybrids containing fusions of the Rep proteins to green fluorescent protein (GFP), we show here that fluorescence from GFP-Rep1p or GFP-Rep2p is almost exclusively localized in the nucleus in a cir ؉ strain. Nuclear localization of GFP-Rep1p and GFP-Rep2p, though discernible, is less efficient in a cir 0 host. GFP-Rep2p or GFP-Rep1p is able to promote the stability of a 2m circle-derived plasmid harboring REP1 or REP2, respectively, in a cir

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Direct simulation of yeast 2-μm circle plasmid amplification

Cited by 6 publications

References 16 publications

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

The 2μm Plasmid Stability System: Analyses of the Interactions among Plasmid- and Host-Encoded Components

The 2microm-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus

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