Distinct classes of mutations in REP1 that affect maintenance of the 2μm plasmid.
According to the current model for 2μm circle partitioning, mutations that adversely affect the DNA-protein or protein-protein interactions within the Rep-STB
system must interfere with plasmid stability. We tested a subset of these predictions by site-directed mutagenesis of the Rep1 protein. The choice of Rep1p over Rep2p for this analysis was dictated by the higher degree of amino acid conservation for the former among yeast plasmids that resemble the 2μm circle in structural and functional organization (27
). Mutations were targeted to invariant or highly conserved amino acids. A subset of the generated Rep1p mutants was analyzed for interactions with Rep2p and STB
As described in previous work (1
), we employed a dihybrid assay to test the interaction between Rep1p mutants and Rep2p, with LEU2
used as the reporter gene. A positive interaction was declared for colony growth in medium lacking leucine in the presence of galactose but not dextrose (7
). Similarly, we used a monohybrid test to examine the interaction between Rep1p mutants and STB
). A positive interaction was inferred from the induced expression of the HIS3
reporter gene and the consequent resistance of the tester strain to high levels of 3-aminotriazole, an inhibitor of the His3 protein. The data from the two types of interaction assays are assembled in Fig. .
FIG. 1. Interaction of Rep1p variants with Rep2p and STB and consequences in plasmid maintenance. (A) Dihybrid assays were performed with strain EGY48 (Table ) and a LEU2 reporter as described previously (28). The wild-type Rep1 protein (C2) (more ...)
Of the 19 Rep1p mutants analyzed, 5 did not yield a detectable interaction with Rep2p (columns 1 to 3, 6 and 16 in Fig. ), whereas 4 had an extremely weak interaction (Fig. , columns 10, 11, 13, and 17). Consistent with the results from previous deletion analyses (22
), four of the mutations responsible for the former phenotype were clustered within the amino-terminal 100 amino acids of Rep1p. Five of the Rep1p mutants did not yield a positive interaction with STB
(Fig. , columns 1, 8, 14, 18, and 19). One mutation, T32K, led to the loss of both Rep2p and STB
interactions (Fig. , columns 1). The expression levels of the mutants that tested negative in either of the two assays were comparable to that of wild-type Rep1p (Fig. , bottom panels).
The combined results from Fig. , together with the performance of individual mutants in a plasmid stability assay, are summarized in Fig. . Each of the Rep1p variants that did not bind to STB (class I), Rep2p (class II), or both STB and Rep2p (class III) failed to support the stable maintenance of a 2μm circle-derived reporter plasmid. In contrast, Rep1p variants that provided plasmid stabilities comparable to that of wild-type Rep1p (designated the “+” class) were positive for both Rep2p and STB interactions. Note that the Rep1 mutants were assayed for plasmid maintenance as GFP fusions and showed normal nuclear localization, as determined by fluorescence microscopy (data not shown).
The results described above provide experimental support for two of the interactions, Rep1p-Rep2p and Rep1p-STB
, being essential for normal plasmid segregation, as is implicit in the partitioning model. The possible functional relevance of the other interactions, Rep1p-Rep1p, Rep2p-Rep2p, and Rep2p-STB
, in plasmid stability remains to be verified. It should be noted, though, that none of the Rep1p mutants used for the experiments described below were defective for interacting with wild-type Rep1p, as verified by in vivo dihybrid assays. For a subset of these mutants, the in vivo results were confirmed by in vitro GST pull-down assays (Fig. S1 in the supplemental material). Furthermore, the self-interaction of Rep2p was also unaffected, since it is not dependent on Rep1p (1
Rep1 mutations and recruitment of the cohesin complex to the STB locus.
We demonstrated earlier that the yeast cohesin complex specifically associates with the STB
locus in a Rep1p- and Rep2p-dependent fashion (14
). We also observed (in unpublished dihybrid experiments) that Rep1p or Rep2p can interact with the cohesin subunit Mcd1p. Since the tester strain contained an endogenous 2μm circle ([cir]+
), it is not clear whether, for either protein, this interaction is dependent on the presence of its partner. Furthermore, we do not know whether the interaction is direct or mediated through one of the other cohesin subunits or a bridging protein extraneous to the cohesin complex. Based on several pieces of circumstantial evidence, we have suggested that cohesin may play functionally similar roles in the chromosomal and 2μm plasmid segregation pathways. If this is true, at least a subset of the Rep1 mutations that adversely affect plasmid partitioning is likely to abolish or interfere with cohesin recruitment to STB
The association between cohesin and STB
DNA was probed in exponentially growing yeast cells by ChIP assays using antibodies directed to a hemagglutinin (HA)-tagged Mcd1 protein (14
) (Fig. ). Similarly, the presence or absence at STB
of Rep1p (or a mutant derived from it) and Rep2p was also monitored by ChIP assays employing antibodies raised against the wild-type Rep proteins. The results obtained with cells expressing native Rep1p, a representative Rep1p mutant belonging to class I (no STB
interaction) or class II (no Rep2p interaction), and the only class III mutant (neither STB
nor Rep2p interaction) are displayed in Fig. . Wild-type Rep1p and its variants were expressed in a [cir]0
host strain (cured of the 2μm plasmid) from the inducible GAL10
promoter. A 2μm circle-derived plasmid expressing Rep2p served as a reporter for the presence of cohesin at STB
. As expected from prior observations, an Mcd1p-STB
association was readily observed in the presence of native Rep1p (Fig. , lane 2, top row). In contrast, in the presence of Rep1p(Y43A), Rep1p(K297Q), or Rep1p(T32K), this association was either extremely weak (Fig. , top rows, lanes 2) or undetectable (Fig. , top row, lane 2). Consistent with the interaction phenotypes of these mutants (Fig. ), Rep1p(Y43A) (class II) was associated with STB
, as was native Rep1p (Fig. , top rows, compare lanes 5); Rep1p(K297Q) (class I) and Rep1p(T32K) (class III) were not (Fig. , top rows, compare lanes 5). The Rep1p mutations had no effect on the association between Rep2p and STB
(Fig. , top rows, lanes 8). The control assays showed that Mcd1p was associated with one of the cognate sites for cohesin on chromosome V regardless of the Rep1p status of the cells (Fig. , bottom rows, lanes 2). As expected, there was no binding of Rep1p (or its variants) or Rep2p to the chromosomal cohesin binding site (Fig. , bottom rows, lanes 5 and 8).
FIG. 2. Association of Mcd1p with STB in the presence of wild-type Rep1p or its variants. ChIP assays were performed with a [cir]0 strain (lacking endogenous 2μm circles) in which the MCD1 gene was tagged with three copies of the HA tag. The tag was present (more ...)
The above results, in conjunction with previous observations (14
), are consistent with the hypothesis that one critical role for the Rep proteins in plasmid partitioning is the recruitment of cohesin to STB
. Furthermore, both Rep1p-Rep2p and Rep1p-STB
interactions must be satisfied simultaneously for this recruitment to occur. An earlier analysis had shown that Rep1p by itself can associate with STB
even in the absence of Rep2p, and vice versa (28
). In principle, a class I Rep1p mutant, by virtue of its normal interaction with Rep2p, may be indirectly recruited to STB
to establish the Rep1p-Rep2p-STB
ternary complex. The outcome from the present experiments contrasts with this expectation and explains why the class II Rep1p mutants are unable to assist the loading of cohesin at STB
Cycling of Rep proteins at STB during the G1-S interval may ensure replication-dependent plasmid-cohesin association and coordination with chromosomal cohesin recruitment.
A possible role for cohesin in the pairing of replicated plasmid clusters (similar to the pairing of sister chromatids) is suggested by the finding that, during the normal cell cycle, the timing of cohesin association and dissociation is the same for the 2μm plasmid and the chromosomes (14
). Furthermore, as is the situation with sister chromatids, a lack of cohesin disassembly due to a noncleavable form of Mcd1p causes the cells to arrest in a large budded state with a single plasmid cluster rather than two separated clusters. One plausible scenario suggested by these data, by analogy to chromosomes, is that the plasmid is primed for cohesin association only during its replication phase. However, when Mcd1p is expressed inappropriately in G1
, it is bound to the STB
locus but not to chromosomal sites, as inferred from chromosome spread assays and substantiated by ChIP analyses (14
). Although cohesin binding to STB
does not necessarily mean the establishment of cohesion between plasmids, the replication independence of Mcd1p binding raises some concern regarding its relevance to partitioning. An important question is whether, during each cell cycle, there is some mechanism for initiating a fresh round of replication-coupled cohesin recruitment at STB
. In this case, even if a fortuitous plasmid-cohesin association were to occur prior to replication, it would be subsequently overridden.
Previous experiments showed that the binding of Mcd1p to STB
reflects that of the whole cohesin complex (14
). When either Smc1p or Smc3p harbors a Ts
mutation, the G1
-expressed Mcd1p protein is not recruited to STB
at the nonpermissive temperature. We wanted to know whether, like normal cohesin-STB
association, that imposed in G1
is also mediated by the Rep proteins. The ChIP assays shown in Fig. were performed in G1
-arrested (and galactose-induced) cells expressing Myc-tagged Mcd1p from the GAL1
promoter. In the presence of wild-type Rep1 and Rep2 proteins, Mcd1p was associated with STB
, but not with the chromosome V binding site (Fig. , lane 2). The Rep1 and Rep2 proteins were also associated with STB
in these cells (Fig. , lanes 5 and 8). In cells expressing a class I Rep1p mutant, Rep1p(K297Q), Mcd1p was not detected at STB
(Fig. , lane 11). As expected, the mutant Rep1p was not present at STB
(Fig. , lane 14), whereas Rep2p was (Fig. , lane 17). Similar experiments showed that G1
-expressed Mcd1p was not associated with STB
when the Rep1 protein contained a class II (Y34A) or class III (T32K) mutation (Fig. S2 in the supplemental material). The mutant Rep1p protein was present at STB
in the former case but not in the latter. Neither mutation had any effect on the Rep2p-STB
association. Thus, even the untimely enlistment of cohesin by the plasmid during G1
is dependent on the functional interactions of Rep1p with Rep2p and STB
FIG. 3. Association of G1-expressed Mcd1p with STB: cell cycle dependence of Rep1p, Rep2p, Mcd1p, and Smc1p association with STB. (A) ChIP analyses were performed with [cir]0 cells arrested in G1 with α factor in the presence of galactose. The tagged (more ...)
We next followed the patterns of occupancy of STB by Rep1p and Rep2p as a function of cell cycle progression in cells grown in dextrose. Simultaneously, the association of Mcd1p, expressed from its native promoter in an epitope-tagged form, with STB as well as with its chromosome V binding site was also monitored. Both Rep1p and Rep2p were associated with STB at the time of release from alpha factor (Fig. , 0 min). As expected, Mcd1p was absent from STB and chromosome V prior to the onset of S phase (Fig. , 0 to 30 min). Strikingly, during the window between the late G1 and early S phases (Fig. , 15 to 30 min), the Rep proteins were cleared from STB. They then reassociated with STB at the same time that an Mcd1p-STB or Mcd1p-chromosome V association was established (Fig. , 30 min). The Rep proteins persisted at STB after cohesin disassembly from STB and chromosome V and into the G1 phase of the subsequent cell cycle (Fig. , 90 to 120 min). The process of dissociation and reassociation was then repeated (Fig. , 120 to 135 min).
The choice of dextrose as the carbon source for the assays depicted in Fig. made them roughly comparable to previously published cell cycle analyses on the stage-dependent acquisition of Mcd1p (or cohesin) by the 2μm plasmid (14
). Results from similar experiments performed in the presence of galactose (with Mcd1p overexpressed continuously during the cell cycle from the GAL
promoter) corroborated the data in Fig. regarding the cycling of Rep proteins at STB
(Fig. S2 in the supplemental material). However, the timing of the DNA-protein association and dissociation steps of interest was different in the two analyses, as was expected because of the increased duration of the cell cycle and the inappropriate expression of Mcd1p during growth in galactose.
As explained below, the ChIP and FACS data shown in Fig. are mutually concordant, even though the FACS patterns from the two cell cycles are not as well matched as the ChIP patterns. One likely contributing factor to this discrepancy was the relaxation in the degree of synchrony as cells passaged from the first to the second cell cycle. Recovery from pheromone arrest might also introduce subtle timing differences between the two cycles, at least in their early phases.
The absence of Mcd1p and the presence of the Rep proteins at STB at 105 min (with most cells displaying 2× DNA content [G2/M] and only a minority having 1× DNA content [G1]) and at 0 min (with almost the entire population being in G1) were not contradictory. Presumably, anaphase had been triggered in the 2× class of cells, causing Mcd1p cleavage and the dissociation of the cohesin complex. Similarly, the 15-min population contained cells set to exit from G1 (note that S phase was established within 30 min), while the 120-min population consisted primarily of late G1 cells, with some entry into S phase indicated by the trailing shoulder of the G1 peak. Combining this information with the ChIP data, the transient exclusion of the Rep proteins from STB may be assigned to the time span straddling the end of G1 and the beginning of S phase. The association of STB with Rep1p, Rep2p, and Mcd1p in the 30-min and 135-min samples signifies that, despite quantitative differences, these populations were qualitatively similar in harboring mostly S phase-to-pre-anaphase cells.
ChIP analyses analogous to those shown in Fig. , performed by targeting HA-tagged Smc1p and native Rep proteins, showed that all three proteins occupied STB
and exited from it at the same time points. The data for Rep1p and Smc1p are presented in Fig. (rows 1 and 2); the pattern for Rep2p was the same as that for Rep1p (data not shown). Note also that the brief exit of Smc1p during the 0- to 30-min and 90- to 120-min intervals was specific to STB
(Fig. , row 2) and did not occur at the chromosome V site (Fig. , row 3). Furthermore, Smc1p was detected at STB
by the ChIP assay only in a [cir]+
strain, not in a [cir]0
strain (Fig. , top row, compare lanes 2 and 5). The association between Smc1p and the 2μm plasmid was specific to STB
, and no interaction with DNA regions within the FLP
, or REP2
genes was detected (results not shown). Together, these data suggest that the Rep proteins likely mediate the recruitment of Mcd1p via the Smc proteins. Note that Smc1p and Smc3p are present throughout the cell cycle, whereas the expression of Mcd1p only occurs close to the onset of S phase (24
In principle, the observed cycling of the Rep proteins at the STB
locus guarantees that the 2μm plasmid only acquires cohesin during each cell cycle in a replication-dependent manner and in coordination with the chromosomes, as would be consistent with the currently entertained partitioning models (14
). In addition, the Rep1 and Rep2 proteins appear to constitute the cohesin loading complex for the plasmid, as inferred from the absence of Smc1p at the STB
locus of a reporter in the [cir]0
background (hence lacking Rep1p and Rep2p) as well as the almost perfect match between the Rep proteins and Smc1p in their patterns of STB
association during the cell cycle. In the case of chromosomes, the Smc1 and Smc3 proteins are associated only with cohesin binding sites, and this association is mediated by the Scc2p/Scc4p cohesin loading complex (5
The association between cohesin and STB is blocked in the rsc2Δ background.
A recent discovery by Wong et al. (34
) revealed a strong correlation between the nucleosome architecture at STB
and the stable propagation of the 2μm plasmid. In the absence of the Rsc2 protein, a component of one of the nucleosome remodeling complexes in yeast, there is a high rate of loss of the 2μm circle (34
). Concomitantly, there is a finite alteration of the chromatin structure of the STB
region. Is the plasmid instability in the RSC2
deletion background possibly brought on by a lack of cohesin recruitment to STB
The results from ChIP assays performed with an rsc2Δ yeast strain and an isogenic wild-type counterpart, both containing a selectively maintained 2μm plasmid derivative [cir]+, to monitor the Mcd1p association with STB are shown in Fig. . Whereas Mcd1p was detected at STB and chromosome V in the wild-type strain (Fig. , lane 2), it was absent from STB in the rsc2Δ strain (Fig. , top row, lane 5). In contrast, the deletion had no apparent effect on the association of Mcd1p with chromosome V (Fig. , bottom row, lane 5).
FIG. 4. Association of Mcd1p or the Rep proteins with STB in RSC2 and RSC1 deletion strains. (A, C, D, and F) Immunoprecipitations were done with the indicated antibodies in a [cir]+ wild-type strain or its rsc2Δ and rsc1Δ derivatives (more ...)
The ChIP results were then verified by a monohybrid assay performed with a chromosomally integrated copy of the STB DNA (Fig. ). In conformity with the data for Fig. , Mcd1p interacted with STB in the wild-type strain (Fig. , lane 2) but not with that in the rsc2Δ strain (Fig. , lane 4).
In contrast to the deletion of RSC2
, the deletion of RSC1
did not have any obvious deleterious effect on the association of Mcd1p with STB
, as determined by a ChIP assay (Fig. , lane 2). These data are consistent with the fact that, unlike the rsc2
Δ mutation, an rsc1
Δ mutation does not lead to an elevated loss of the 2μm plasmid (34
The above findings suggest a model in which the Rsc2 protein facilitates 2μm circle stability by preserving the chromatin structure of STB in a state that is conducive for recruiting cohesin. When this functional architecture of the locus is altered due to the lack of one of the RSC nucleosome remodeling complexes, STB is rendered incompetent in acquiring cohesin. As a result, cohesin-mediated equal partitioning of the plasmid becomes impossible.
Absence of Rsc2 protein affects binding of Rep1p but not Rep2p to STB.
In principle, the exclusion of cohesin from STB in the rsc2Δ strain may result from the inability of Rep1p, Rep2p, or both to bind STB in its altered chromatin state. As shown in this study (Fig. ), a mutation in Rep1p that eliminates STB binding also blocks STB-cohesin association.
We therefore monitored the effect of the RSC2 deletion on the occupancy of STB by Rep1p and Rep2p by using ChIP assays (Fig. ) as well as monohybrid (Fig. ) assays. As expected, in the wild-type background, both the Rep1 and Rep2 proteins bound to STB (Fig. , lanes 2 and 5, and 4E, lanes 2 and 3). In the absence of Rsc2p, the association between Rep1p and STB was abolished (Fig. , lane 8, and 4E, lane 5). On the other hand, Rep2p binding to STB was unaffected by the rsc2Δ mutant (Fig. , lane 11, and 4E, lane 6).
In the case of the rsc1Δ mutant, both Rep1p and Rep2p were found to be present at STB, when analyzed by ChIP (Fig. , lanes 2 and 5). This was expected since the deletion of RSC1 did not affect Mcd1p (or cohesin, by inference) recruitment to STB (Fig. ).
The data from Fig. suggest that the nucleosome organization at STB promoted by the Rsc2p-containing remodeling complex is specific to the recruitment of Rep1p. Rep2p can occupy STB even when the latter is in a nonfunctional chromatin state induced by the absence of Rsc2p. The RSC2 deletion and mutations of Rep1p that eliminate its interaction with STB (class I) (Fig. ) are therefore functionally equivalent in destabilizing the 2μm plasmid. According to our current thinking, their downstream effect of preventing plasmid-cohesin association is the likely cause for plasmid loss. At this time, we cannot rule out the possible alternative that the interaction between Rep1p and STB, which is abolished in the absence of Rsc2p, is what is essential for the nucleosome organization at STB.
Blocking cohesin-STB association without affecting Rep-STB interactions causes 2μm circle missegregation.
We previously showed that a temperature-sensitive mutation in a component of the cohesin complex (Smc1p or Smc3p) blocks its association with STB
, as well as with chromosomal cohesin binding sites, at the nonpermissive temperature (14
). Do the Rep1 and Rep2 proteins stay associated with STB
under restrictive conditions? Furthermore, how is 2μm circle segregation affected by the mutation? The answers to these questions, displayed in Fig. , have an important bearing on the suspected role of cohesin in plasmid partitioning.
FIG. 5. Absence of Mcd1p-STB association and high level of plasmid missegregation in the smc1-2 strain at a nonpermissive temperature. (A to D) Association of Mcd1p, Smc1p, or the Rep proteins with STB as monitored by ChIP in exponentially growing cells at 26°C (more ...)
ChIP results for the smc1-2 (Ts) strain, obtained by using antibodies directed to the Mcd1 protein, confirmed our earlier finding that cohesin could not be detected at STB or at the chromosome V binding site in cells arrested at 37°C but was present at these locales in cells growing at 26°C (Fig. , compare lanes 2 and 5). When ChIP was performed with the mutant Smc1p being tagged with Myc, occupancy of STB by the protein was detected at the permissive temperature (Fig. , lane 2) but not at the nonpermissive temperature (Fig. , lane 5). Assays with Rep protein antibodies showed Rep1p and Rep2p to be associated with STB at both temperatures (Fig. , lanes 2 and 5).
In one explanation of the above data, it is the whole cohesin complex, containing Mcd1p as well as Smc1p, that binds to STB
. When preassembly of the complex is prevented, individual subunits can no longer bind to STB
. In a second explanation, during the assembly of the cohesin complex at STB
, the association of Smc1p with STB
precedes that of Mcd1p. The latter explanation is favored by the data in Fig. showing that Smc1p is associated with STB
, even though Mcd1p expression is not turned on until later in the cell cycle (5
). In either case, the recruitment of cohesin to the 2μm plasmid can be interrupted by a mutation in one of its subunits, while apparently normal interactions between the Rep proteins and the STB
DNA are retained.
We next examined the pattern of plasmid partitioning in the smc1-2 strain under permissive and restrictive conditions, as indicated schematically in Fig. . Cells from a log-phase culture grown at 26°C were first arrested in G1 by use of alpha factor and were then released into a pheromone-free medium at 26 or 37°C. For the 26°C population, the plasmid partitioning data were derived from large budded cells (constituting the major subset of cells) present 1 h after the cell cycle restart. For the 37°C population, the corresponding values were obtained from cells arrested in the large budded state (>80% of the population) after 2 h at the shifted temperature. In estimating the values for the two cell types depicted in Fig. , the criterion for normal segregation (class I) was a rough equivalence between the two cell compartments in chromosome content as well as plasmid content, as revealed by DAPI staining and plasmid-associated fluorescence, respectively. For the plasmid, any inequity in partitioning was additionally displayed by the difference in the number of fluorescent foci between the clusters present in the two compartments. The most prominent missegregation phenotype at 37°C was one in which the plasmid and the chromosomes missegregated in a proportional manner. In this group of cells (class II), the two cell compartments clearly displayed nonuniformity in plasmid-associated fluorescence as well as DAPI fluorescence. However, the cell compartment with a higher chromosome content also had a higher plasmid content. The ratio of class II cells to the sum of class I and class II cells, called the aberrant segregation index, rose from <2% at 26°C to approximately 70% at 37°C.
The above analysis demonstrates that gross plasmid missegregation can be effected by obstructing cohesin recruitment to STB while leaving interactions between STB and the Rep proteins unaffected. Given this result, the incompetence of the class I to III Rep1p mutants in plasmid maintenance is most readily explained by their common inability to mediate the cohesin-STB association.