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

The stable inheritance of the 2μm 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 2μm circle-derived plasmid shows relatively poor stability.     

The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning

Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres (TELs) and centromeres (CENs) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TELs (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus (STB) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.     

Temporal sequence and cell cycle cues in the assembly of host factors at the yeast 2 micron plasmid partitioning locus

The Saccharomyces cerevisiae 2 micron plasmid exemplifies a benign but selfish genome, whose stability approaches that of the chromosomes of its host. The plasmid partitioning locus STB (stability locus) displays certain functional analogies with centromeres along with critical distinctions, a significant one being the absence of the kinetochore complex at STB. The remodels the structure of chromatin (RSC) chromatin remodeling complex, the nuclear motor Kip1, the histone H3 variant Cse4 and the cohesin complex associate with both loci. These factors appear to contribute to plasmid segregation either directly or indirectly through their roles in chromosome segregation. Assembly and disassembly of the plasmid-coded partitioning proteins Rep1 and Rep2 and host factors at STB follow a temporal hierarchy during the cell cycle. Assembly is initiated by STB association of [Rsc8-Rsc58], followed by [Rep1-Rep2-Kip1] and [Cse4-Rsc2-Sth1] recruitment, and culminates in cohesin assembly. Disassembly starts with dissociation of RSC components, is followed by cohesin disassembly and Cse4 exit during anaphase and late telophase, respectively. [Rep1-Rep2-Kip1] persists through G1 of the ensuing cell cycle. The de novo assembly of the ‘partitioning complex’ is cued by the innate cell cycle clock and is dependent on DNA replication. Shared functional attributes of STB and centromere (CEN) are consistent with a potential evolutionary link between them.     

Microtubule‐associated proteins,Bik1 and Bim1, are required for faithful partitioning of the endogenous 2 micron plasmids in budding yeast

The 2 μ plasmid of budding yeast shows high mitotic stability similar to that of chromosomes by using its self‐encoded systems, namely partitioning and amplification. The partitioning system consists of the plasmid‐borne proteins Rep1, Rep2 and a cis‐acting locus STB that, along with several host factors, ensures efficient segregation of the plasmid. The plasmids show high stability as they presumably co‐segregate with chromosomes through utilization of various host factors. To acquire these host factors, the plasmids are thought to localize to a certain sub‐nuclear locale probably assisted by the motor protein, Kip1 and microtubules. Here, we show that the microtubule‐associated proteins Bik1 and Bim1 are also important host factors in this process, perhaps by acting as an adapter between the plasmid and the motor and thus helping to anchor the plasmid to microtubules. Abrogation of Kip1 recruitment at STB in the absence of Bik1 argues for its function at STB upstream of Kip1. Consistent with this, both Bik1 and Bim1 associate with plasmids without any assistance from the Rep proteins. As observed earlier with other host factors, lack of Bik1 or Bim1 also causes a cohesion defect between sister plasmids leading to plasmid missegregation.     

The selfish yeast plasmid uses the nuclear motor Kip1p but not Cin8p for its localization and equal segregation

The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1p association with the plasmid partitioning locus STB are consistent with Kip1p being an authentic component of the plasmid partitioning complex. Kip1–STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a “partitioning center” represents yet another facet of the benign parasitism of the yeast plasmid.     

A selfish DNA element engages a meiosis-specific motor and telomeres for germ-line propagation

The chromosome-like mitotic stability of the yeast 2 micron plasmid is conferred by the plasmid proteins Rep1-Rep2 and the cis-acting locus STB, likely by promoting plasmid-chromosome association and segregation by hitchhiking. Our analysis reveals that stable plasmid segregation during meiosis requires the bouquet proteins Ndj1 and Csm4. Plasmid relocalization from the nuclear interior in mitotic cells to the periphery at or proximal to telomeres rises from early meiosis to pachytene. Analogous to chromosomes, the plasmid undergoes Csm4- and Ndj1-dependent rapid prophase movements with speeds comparable to those of telomeres. Lack of Ndj1 partially disrupts plasmid–telomere association without affecting plasmid colocalization with the telomere-binding protein Rap1. The plasmid appears to engage a meiosis-specific motor that orchestrates telomere-led chromosome movements for its telomere-associated segregation during meiosis I. This hitherto uncharacterized mode of germ-line transmission by a selfish genetic element signifies a mechanistic variation within the shared theme of chromosome-coupled plasmid segregation during mitosis and meiosis.     

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

The efficient partitioning of the 2microm 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 2microm circle-derived plasmid harboring REP1 or REP2, respectively, in a cir(0) background. Under these conditions, fluorescence from GFP-Rep2p or GFP-Rep1p is concentrated within the nucleus, as is the case in cir+ cells. This characteristic nuclear accumulation is not dependent on the expression of the FLP or RAF1 gene of the 2microm circle. Nuclear colocalization of Rep1p and Rep2p is consistent with the hypothesis that the two proteins directly or indirectly interact to form a functional bipartite or high-order protein complex. Immunoprecipitation experiments as well as baiting assays using GST-Rep hybrid proteins suggest a direct interaction between Rep1p and Rep2p which, in principle, may be modulated by other yeast proteins. Furthermore, these assays provide evidence for Rep1p-Rep1p and Rep2p-Rep2p associations as well. The sum of these interactions may be important in controlling the effective cellular concentration of the Rep1p-Rep2p complex.     

Mutations in a partitioning protein and altered chromatin structure at the partitioning locus prevent cohesin recruitment by the Saccharomyces cerevisiae plasmid and cause plasmid missegregation

The 2 microm circle is a highly persistent "selfish" DNA element resident in the Saccharomyces cerevisiae nucleus whose stability approaches that of the chromosomes. The plasmid partitioning system, consisting of two plasmid-encoded proteins, Rep1p and Rep2p, and a cis-acting locus, STB, apparently feeds into the chromosome segregation pathway. The Rep proteins assist the recruitment of the yeast cohesin complex to STB during the S phase, presumably to apportion the replicated plasmid molecules equally to daughter cells. The DNA-protein and protein-protein interactions of the partitioning system, as well as the chromatin organization at STB, are important for cohesin recruitment. Rep1p variants that are incompetent in binding to Rep2p, STB, or both fail to assist the assembly of the cohesin complex at STB and are nonfunctional in plasmid maintenance. Preventing the cohesin-STB association without impeding Rep1p-Rep2p-STB interactions also causes plasmid missegregation. During the yeast cell cycle, the Rep1p and Rep2p proteins are expelled from STB during a short interval between the late G(1) and early S phases. This dissociation and reassociation event ensures that cohesin loading at STB is replication dependent and is coordinated with chromosomal cohesin recruitment. In an rsc2 Delta yeast strain lacking a specific chromatin remodeling complex and exhibiting a high degree of plasmid loss, neither Rep1p nor the cohesin complex can be recruited to STB. The phenotypes of the Rep1p mutations and of the rsc2 Delta mutant are consistent with the role of cohesin in plasmid partitioning being analogous to that in chromosome partitioning.     

Partitioning of the 2-microm circle plasmid of Saccharomyces cerevisiae. Functional coordination with chromosome segregation and plasmid-encoded rep protein distribution

The efficient partitioning of the 2-microm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-microm-derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-microm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.     

Two Rep Genes in Small Cryptic Plasmid pKST21 of Escherichia coli

The complete nucleotide sequence of a small cryptic plasmid pKST21 from Escherichia coli was determined. This plasmid is 1,460 bp long with an overall GC content of 51 %. Based on sequence analysis, the presence of two segments with different average GC density was observed. The segment with higher GC content revealed 98–90 % similarity to several small plasmids of E. coli and to pCR1 from Gram-positive Corynebacterium renale. Plasmid pKST21 possesses two conversely oriented open reading frames encoding proteins with a high degree of amino acid identity to Rep proteins involved in replication. ORF1 encodes replication protein similar to RepA protein of Bartonella tribocorum or Bacillus cereus plasmids or to the putative plasmid Rep protein from ecologically close Selenomonas ruminantium. ORF2 similarly encodes a replication protein, which shares 97 % homology with Rep protein from C. renale. Genetic diversity observed in plasmid pKST21 indicates a mosaic structure of the plasmid with different segments acquired from different sources. Deletion analysis showed that both fragments carrying the repA and repB genes are necessary for the replication of pKST21 in E. coli. The presence of plasmid with the same gene composition was revealed in 14 % of tested E. coli isolates from the rumen of sheep. All these strains produced identical ERIC-PCR profiles indicating isogenic origin of the strain and lack of horizontal gene transfer of pKST21 plasmid.     

The 2 micron plasmid purloins the yeast cohesin complex: a mechanism for coupling plasmid partitioning and chromosome segregation?

The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes.     

The Rep20 Replication Initiator From the pAG20 Plasmid of Acetobacter aceti

In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM3610 strain, the Rep20 protein was characterized as a main replication initiator. The pAG20 plasmid origin was localized in the vicinity of the rep20 gene and contained two 21-nucleotide-long iteron sequences, two 13-nucleotide-long direct repeats, and a DnaA-binding site. Electrophoretic mobility shift assay and nonradioactive fragment analysis confirmed that the Rep20 protein interacted with two direct repeats (5′-TCCAAATTTGGAT′-3′) and their requirement during plasmid replication was verified by mutagenesis. Although the association could not be validated of the DnaA protein of from the host cells of Escherichia coli with the plasmid-encoded replication initiator that usually occurs during replication initiation, Rep20 was able to form dimeric structures by which it could bind the sequence of the rep20 gene and autoregulate its own expression. Targeted mutagenesis of the Rep20 protein revealed the importance of the third α-helix and ⁶³Lys, specifically during DNA binding. The second, closely adjacent β-sheet also took part in this process in which ⁵²Asn played a significant role.     

Functional domains of yeast plasmid-encoded Rep proteins

Both of the Saccharomyces cerevisiae 2 microm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2 microm-based stability vector with rep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2 microm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.     

Topoisomerase activity of the hyperthermophilic replication initiator protein Rep75

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via the rolling circle mechanism. pGT5 encodes the replication initiator protein Rep75 that exhibits a nicking–closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin (dso) sequence. Some mesophilic Rep proteins present site-specific DNA topoisomerase-like activity on a negatively supercoiled plasmid harbouring the dso. We report here that Rep75 also exhibits topoisomerase activity on a negatively supercoiled DNA substrate. This DNA topoisomerase-like activity is dependent on the amino acids involved in NC activity of Rep75. However, in contrast with mesophilic Rep proteins, Rep75 topoisomerase activity is not dso dependent. Moreover, although pGT5 is known to be relaxed in vivo, Rep75 was not able to act on a relaxed plasmid in vitro, whether or not it contained the dso.     

Distinct SUMO Ligases Cooperate with Esc2 and Slx5 to Suppress Duplication-Mediated Genome Rearrangements

Suppression of duplication-mediated gross chromosomal rearrangements (GCRs) is essential to maintain genome integrity in eukaryotes. Here we report that SUMO ligase Mms21 has a strong role in suppressing GCRs in Saccharomyces cerevisiae, while Siz1 and Siz2 have weaker and partially redundant roles. Understanding the functions of these enzymes has been hampered by a paucity of knowledge of their substrate specificity in vivo. Using a new quantitative SUMO-proteomics technology, we found that Siz1 and Siz2 redundantly control the abundances of most sumoylated substrates, while Mms21 more specifically regulates sumoylation of RNA polymerase-I and the SMC-family proteins. Interestingly, Esc2, a SUMO-like domain-containing protein, specifically promotes the accumulation of sumoylated Mms21-specific substrates and functions with Mms21 to suppress GCRs. On the other hand, the Slx5-Slx8 complex, a SUMO-targeted ubiquitin ligase, suppresses the accumulation of sumoylated Mms21-specific substrates. Thus, distinct SUMO ligases work in concert with Esc2 and Slx5-Slx8 to control substrate specificity and sumoylation homeostasis to prevent GCRs.     

A novel role for the mitotic spindle during DNA segregation in yeast: promoting 2 microm plasmid-cohesin association

The 2 microm circle plasmid in Saccharomyces cerevisiae is a model for a stable, high-copy-number, extrachromosomal "selfish" DNA element. By combining a partitioning system and an amplification system, the plasmid ensures its stable propagation and copy number maintenance, even though it does not provide any selective advantage to its host. Recent evidence suggests that the partitioning system couples plasmid segregation to chromosome segregation. We now demonstrate an unexpected and unconventional role for the mitotic spindle in the plasmid-partitioning pathway. The spindle specifies the nuclear address of the 2 microm circle and promotes recruitment of the cohesin complex to the plasmid-partitioning locus STB. Only the nuclear microtubules, and not the cytoplasmic ones, are required for loading cohesin at STB. In cells recovering from nocodazole-induced spindle depolymerization and G(2)/M arrest, cohesin-STB association can be established coincident with spindle restoration. This postreplication recruitment of cohesin is not functional in equipartitioning. However, normally acquired cohesin can be inactivated after replication without causing plasmid missegregation. In the mtw1-1 mutant yeast strain, the plasmid cosegregates with the spindle and the spindle-associated chromosomes; by contrast, a substantial number of the chromosomes are not associated with the spindle. These results are consistent with a model in which the spindle promotes plasmid segregation in a chromosome-linked fashion.