Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.

Author Summary
Replication termination is the final step of the replication process, where the two replication forks converge and finally merge to form fully replicated sister chromatids. During this process topological strain in the form of DNA overwinding is generated between forks, and if not removed this strain will inhibit replication of the remaining DNA and Introduction During DNA replication termination two replication forks coming from opposite directions merge to form two fully replicated sister chromatids. The process is essential for correct transmission of the genetic information to the next generation, but it has so far attracted little attention in eukaryotes. In E. coli, replication terminates in a region defined by two sets of Ter sites bound by the polar terminator protein, Tus. This protein stops replication forks from one direction, but allows free passage of forks from the opposite direction. The Tus-Ter sites are organized so that they form a trap for replication forks, thereby ensuring termination in a region opposite oriC in the circular E. coli genome [1]. Polar replication fork barriers with a function in replication termination have also been identified in yeast. In S. cerevisiae the rDNA locus holds the Replication Fork Barrier sequence (RFB). This barrier binds the Fob1 protein, which mediates polar fork stalling at RFB, resulting in replication termination in this region [2]. In S. pombe polar replication fork barriers are found both at the rDNA and mating type loci, where replication fork arrest occurs at the termination sites TER1-3 and RFP4 [3] and at the Replication Termination Sequence 1 (RTS1) [4], respectively. At yeast barriers members belonging to the Pif1 family helicases, Rrm3 and Pif1 in S. cerevisiae and Pfh1 in S. pombe, have been demonstrated to play profound roles for fork stalling and fork merging [5,6,7]. 71 termination regions (TERs) have been identified in the S. cerevisiae genome outside the rDNA locus in one of the first large-scale studies performed on this subject in eukaryotes [8]. A common theme to the sequences at the identified TERs was that they contained fork pausing elements and that the Rrm3 protein assisted fork progression through these zones. Furthermore, DNA topoisomerase II located to the TERs during the S and G2/M phases and prevented DNA breaks and genome rearrangements, suggesting that topoisomerase II plays a role to ensure proper replication termination.
Over the years several studies have implicated topoisomerase II in the final steps of replication. Early studies of the circular SV40 genome and the yeast-borne 2μ plasmid reported incomplete replication with nascent strands containing smaller or larger gaps upon inhibition of topoisomerase II activity [9,10,11,12]. Based on these studies a model was presented, suggesting that positive supercoiling accumulates between converging forks, leading to a rotation of the replisomes and formation of precatenanes behind the forks. As a consequence, genuine catenanes form following termination in the absence of topoisomerase II activity, since precatenanes are exclusively substrates for this enzyme [13,14].
The STR complex, which in S. cerevisiae consists of the Sgs1 RecQ helicase, topoisomerase III (Top3) and the Rmi1 protein, has mainly been studied in relation to its role downstream of homologous recombination (HR) [15,16,17]. Studies have provided evidence that the complex is involved in dissolution of double Holliday Junctions (dHJ) in a non-crossover process [18]. In this process Sgs1 is thought to disrupt local annealing between parental and nascent strands, thereby forming hemicatenanes, which can be decatenated by Top3 [16,18]. However, based on early results demonstrating an interaction between Sgs1 and topoisomerase II (Top2), as well as a chromosomal missegregation phenotype of sgs1Δ cells, components of the STR complex have also been proposed to play a role during late stages of replication [19]. In support of this, Marians and co-workers demonstrated that RecQ and topoisomerase III from E. coli in collaboration with the single-stranded DNA-binding protein SSB performed the resolution of a synthetic termination substrate in vitro [20]. The parental duplex DNA region between two stalled replication forks was here unwound by the RecQ helicase, while topoisomerase III simultaneously decatenated the resulting catenated strands, leading to gapped, but untangled termination regions. In line with this, Hickson and co-workers reported that the BLM-TOP3-RMI1-RMI2 complex localized specifically to ultrafine DNA structures, the socalled anaphase bridges, during M phase in human cells. It has been speculated that these bridges represent late replication intermediates that are resolved by the BLM-TOP3-RMI1-RMI2 complex [21,22,23]. Similarly, Sgs1 and Top3 were found to localize to anaphase bridges in budding yeast [24]. Together, these data indicate that RecQ helicases in concert with topoisomerase III play a role in late stages of replication besides their well-established role in the resolution of recombination structures.
Our aim with the present study has been to investigate if S. cerevisiae Top2 and Sgs1-Top3 act redundantly in vivo to ensure faithful replication termination and resolution of replicating chromatids. Our findings demonstrate that they do, and their redundant function in this process is restricted to the rDNA locus.

Results
Lack of Top2 and Sgs1-Top3 leads to robust checkpoint activation in late S/G2 Replicative stress and perturbations activate the S phase checkpoint pathway, which coordinates replication, repair, and cell cycling [25]. The Rad53 kinase is essential to this pathway and is phosphorylated, when the pathway is activated. If Top2 and the STR complex play redundant functions during final stages of replication, we expect that the absence of Top2 and Sgs1 will cause problems in late S/G2, which may activate Rad53. To test this hypothesis we monitored Rad53 phosphorylation in sgs1Δtop2 ts cells using the In Situ Autophosphorylation (ISA) assay, which takes advantage of the autophosphorylation activity of Rad53, when it has been primed by upstream kinases [26]. As illustrated in the experimental setup presented in Fig 1A, yeast cells were grown at 25°C, synchronized in the G1 phase of the cell cycle with αfactor and released into the S phase, where samples were withdrawn at different time points and processed for checkpoint analyses. Release was at 34°C, the restrictive temperature for top2 ts . In the top2 ts mutant, checkpoint activation was seen 120 minutes after release from αfactor ( Fig 1B) in accordance with the previously identified function of Top2 in chromosome segregation [27,28]. Consistent with this, no checkpoint activation was seen in top2 ts cells treated with nocodazole, which prevents the cells from entering mitosis by inhibiting microtubule polymerization (Fig 1C). Interestingly, the sgs1Δtop2 ts double mutant showed robust checkpoint activation already 60 minutes after release into S phase (Fig 1B). To analyze whether this checkpoint was connected to failure in chromosomal segregation due to lack of Top2, sgs1Δtop2 ts cells were treated with nocodazole. However, the robust checkpoint activation persisted in the presence of nocodazole and was thus independent of chromosome segregation ( Fig 1C). As revealed by FACS analyses the observed checkpoint occurred in late S/G2 after bulk DNA synthesis had taken place.
Most known functions of Sgs1 are mediated through the STR-complex, where Sgs1 acts in concert with Top3 and Rmi1. To investigate if this was the case here, we tested a top2 ts top3 ts Loss of Top2 and Sgs1-Top3 leads to chromosome segregation-independent checkpoint activation. (A) Schematic illustration of the experimental setup. ISA analysis of Rad53 was performed with wt, sgs1Δ, top2 ts , and sgs1Δtop2 ts cells (B), with top2 ts and sgs1Δtop2 ts cells treated with nocodazole (noc) as indicated (C), or with top3 ts and top2 ts top3 ts cells (D). At the indicated time points after cells were released into YPD-media at 34°C, the restrictive temperature for top2 ts (B and C) or at 37°C, a restrictive temperature strain for checkpoint activation. The top2 ts top3 ts cells showed robust checkpoint activation 60 minutes after release into S phase (Fig 1D), illustrating that Sgs1 and Top3 are equally important in cells lacking Top2. Taken together, the data demonstrate that Top2 and components of the STR-complex function in late S/G2 to avoid the accumulation of checkpoint activating structures.
Deletion of FOB1 abolishes checkpoint activation in sgs1Δtop2 ts cells Pulsed-field gel electrophoresis (PFGE) has earlier been used to demonstrate replication abnormalities in yeast, because DNA structures associated with incompletely replicated chromosomes prevent gel entrance [29,30]. We therefore applied this technique to investigate if the checkpoint activating structures observed in sgs1Δtop2 ts cells would affect the fate of the individual chromosomes during replication. EtBr stainings of the PFGs revealed a decrease in the intensity of the individual chromosomal bands 40 minutes after α-factor release (Fig 2A, upper panels), which correlated with active replication for all strains as revealed from the FACS profiles. However, after 60 minutes the intensity of the bands had increased, demonstrating completion of replication and that none of the strains experienced any overall defect in replication. However, in the sgs1Δtop2 ts cells, chromosome XII (chr. XII), which holds the rDNA locus, never re-entered the gel after replication, as confirmed by Southern blotting with an rDNA specific probe (Fig 2A, middle panels). In contrast, chr. II, which was used as a control for the other chromosomes, re-entered the gel (Fig 2A, lower panels). A quantification of the amount of chr. XII relative to chr. II re-entering the gel is shown in Fig 2B. Thus, although bulk DNA synthesis seems to occur without major problems in sgs1Δtop2 ts cells, either Sgs1 or Top2 is required to complete replication of chr. XII.
A major part of budding yeast chr. XII is made up of the rDNA locus, consisting of 100-200 repeats of a 9.1 kb unit holding the 35S and 5S rRNA genes. Each repeat holds a replication origin (ARS) and a replication fork barrier (RFB) sequence (Fig 2C), where the latter forms a unidirectional replication fork block, when bound by the Fob1 protein. This block specifically stalls replication forks coming from the direction of the 5S rRNA gene and inhibits head-on collision between replication and transcription of the 35S rRNA gene [31]. Stalling of the leftward moving fork at RFB also ensures that replication terminates within this region. The rDNA locus differentiates chr. XII from the remaining chromosomes and could thus be an obvious cause to the problems observed with this chromosome in sgs1Δtop2 ts cells. To address if this was the case and if the underlying cause of the checkpoint activation observed in sgs1Δtop2 ts cells was connected to Fob1-mediated unidirectional replication, we investigated the checkpoint response in a sgs1Δtop2 ts fob1Δ triple mutant. Interestingly, the checkpoint signal was reduced to background levels in the triple mutant ( Fig 2D). Thus, checkpoint activation in sgs1Δtop2 ts cells is Fob1-dependent and therefore takes place as a result of events occurring at the rDNA locus.
It is well established that excessive amounts of ssDNA coated with the ssDNA binding protein RPA is a signal for checkpoint activation through the Mec1 kinase [32,33]. To investigate if the Fob1-dependent checkpoint activation observed in sgs1Δtop2 ts cells was related to the formation of DNA structures containing ssDNA, foci analysis were performed with cells having the large subunit of RPA (Rfa1) tagged with CFP and Nop1 (a marker for the nucleolus [34]) for both top2 ts and top3 ts (D), samples were taken and processed for ISA analysis. The positive control (+control) represents ISA analysis on extract from MMS-treated wt cells. Mcm2 was used as a loading control. FACS profiles of samples taken throughout the experiment are shown below each strain. 1C and 2C indicate DNA content in G1 and G2, respectively, and asyn. refers to asynchronously growing cells. doi:10.1371/journal.pgen.1005697.g001 Top2 and Sgs1-Top3 in rDNA Replication Termination The results demonstrated that sgs1Δtop2 ts cells experienced significantly more RPA foci and thus more ssDNA 60-100 minutes after α-factor release relative to wt cells and single mutants, and most of the foci had a perinucleolar localization (S1A Fig Fur-thermore, formation of the ssDNA was Fob1-dependent (S1B Fig). Thus, the Fob1-dependent checkpoint activation observed in sgs1Δtop2 ts cells is associated with a Fob1-dependent formation of ssDNA at the rDNA locus. The data indicate that the checkpoint activating DNA-structures observed in sgs1Δtop2 ts cells contain regions of ssDNA.
Late S/G2 checkpoint activation correlates with appearance of Xstructures at the rDNA locus To further analyze the nature of the checkpoint activating structures formed in the rDNA in sgs1Δtop2 ts cells, we performed Neutral-Neutral two-dimensional (2D) gel electrophoresis with genomic DNA from sgs1Δtop2 ts and control strains ( Fig 3A). The BglII restriction sites used for generation of a DNA fragment with the RFB site centrally located (BglIIB) is shown in Fig 2C, and the migration of the replication structures obtained with this fragment in 2D gels is shown in Fig 3B. 40 minutes after release from α-factor, active rDNA replication occurred in all strains as demonstrated by formation of single and double Y-structures as well as structures generated due to replication fork blockage and convergence at RFB (Fig 3A). 80 minutes after α-factor release replication termination had occurred at most rDNA repeats in wt and single mutants as reflected by the disappearance of the majority of replication intermediates. However, a remarkable accumulation of DNA structures giving rise to a significant X-spike had taken place in sgs1Δtop2 ts cells already 60 minutes after α-factor release, which coincided with the timing of checkpoint activation. Notably, the X-spike included the dot from symmetric X-structures representing forks converging at RFB as well as asymmetric X-structures extending the X-spike half way towards the 2N dot (indicated by the stippled area in Fig 3B). Quantification of the Xspike signal relative to the signal from Y-structures demonstrated an increase in the relative amounts of X-spike over time (Fig 3C), although the total amount of replication structures, including the X-structures, had decreased significantly after 100 minutes ( Fig 3A). In contrast, sgs1Δtop2 ts cells did not show an increase in RFB stalling relative to wt cells as revealed from a quantification of the RFB signal relative to the signal from all Y-structures ( Fig 3D).
To investigate if X-structure formation was restricted to the area around RFB we analyzed the migration of replication structures obtained in the BglIIA fragment (see Fig 2C) covering most of the 35S transcription unit. Asymmetric X-structures were also formed in this fragment with the same timing and Xs/Ys ratio as in the BglIIB fragment ( Fig 3E). In contrast, we did not see an accumulation of X-structures in sgs1Δtop2 ts cells, when replication structures were analyzed in a fragment outside the rDNA, containing the TER102 site on chr. I [8] (S2 Fig). Thus, lack of Sgs1 and Top2 causes an accumulation of X-structures in late S/G2, which seems to be restricted to the rDNA locus.

Different structures account for checkpoint activation and X-spike formation
Our data demonstrate that sgs1Δtop2 ts cells show two strong characteristics, checkpoint activation and formation of X-structures, which are both connected to the rDNA locus. To  investigate if the X-structures were the cause of checkpoint activation we took advantage of the Fob1-dependency of checkpoint activation and analyzed replication structures formed in the sgs1Δtop2 ts fob1Δ triple mutant by 2D gel electrophoresis (Fig 4). Interestingly, the X-spike was still present in both BglII fragments. Thus, in contrast to the checkpoint activation (Fig 2D), all X-structures (except the symmetric X-structure formed due to forks converging at RFB) are formed independent of Fob1. Based on this we conclude that asymmetric X-structures are not responsible for checkpoint activation.
An investigation of chromosome migration in the sgs1Δtop2 ts fob1Δ triple mutant by PFGE furthermore demonstrated that lack of Fob1 in the sgs1Δtop2 ts strain was unable to suppress the migration defect of chr. XII (Fig 4), strongly indicating that the presence of X-structures in the rDNA is responsible for the inability of chr. XII to re-enter the gel after replication.
Taken together, our data are most consistent with a formation of two different DNA structures in sgs1Δtop2 ts cells, a Fob1-dependent structure, which causes checkpoint activation, and a Fob1-independent structure causing the formation of the X-spike and the migration defect of chr. XII.
That checkpoint activation and X-spike formation are caused by different DNA structures was further supported by results obtained from experiments, where we investigated the sensitivity of the different structures to topoisomerase activity. In these experiments we reactivated Top2 in sgs1Δtop2 ts cells (S3B and S3C Fig) and Top2 and Top3 in top2 ts top3 ts cells (S3D and S3E Fig) either before or after checkpoint activation and X-spike formation (25 or 60 minutes after release, respectively). We found that checkpoint activation and X-spike formation were inhibited upon early topoisomerase reactivation in both strains. In contrast, checkpoint activation was fully resistant to late reactivation in both strains, whereas X-spike formation was sensitive, showing a small reduction in the Xs/Ys ratio upon Top2 reactivation in sgs1Δtop2 ts cells and a significant reduction upon reactivation of both topoisomerases in top2 ts top3 ts cells.

Checkpoint activation and formation of X-spikes occur independently of homologous recombination
The rDNA locus has previously been demonstrated to be highly recombinogenic with recombination hot spots located close to the RFB [35]. Furthermore, increased recombination activity has been observed within this locus both in sgs1Δ cells and top2 ts mutants kept at semi-permissive conditions [29,36]. Since HR is visualized as X-structures in 2D gels [37] and since one of the important functions of Sgs1-Top3 is to dissolve dHJs downstream of Rad52-mediated HR [38], we wanted to investigate the relationship between HR and checkpoint activation as well as formation of X-structures in the sgs1Δtop2 ts cells. We therefore deleted RAD52 or RAD51 in the sgs1Δtop2 ts strain to investigate if lack of HR would suppress the checkpoint phenotype of sgs1Δtop2 ts cells (Fig 5A and 5B). Although single and double mutants with rad52Δ ( Fig 5A  and S4 Fig) or rad51Δ (Fig 5B) showed a slight increase in basal checkpoint activation as expected, robust checkpoint activation was observed in the two triple mutants. This demonstrates that the checkpoint activation observed in sgs1Δtop2 ts cells occurs independently of HR. Thus, checkpoint activation does not arise due to a HR structure left unresolved in the absence of the Sgs1-Top3 pathway.
the "top of the X-spike". The stippled area below represents asymmetric X-structures, which make up the "main X-spike" in sgs1Δtop2 ts cells. (C) Quantification of Xs to Ys at the indicated time points, where the Xs/Ys obtained at time point 0 was set to 1. Error bars represent STDEV from two to four independent experiments. (D) Quantification of the RFB signal relative to all Ys at the indicated time points, where the ratio at time point 0 was set to 1. Error bars represent STDEV from three to four independent experiments. (E) Genomic DNA was isolated from sgs1Δtop2 ts cells and treated as in (A) except that probe P2 recognizing the BglIIA fragment (Fig 2C) was used in the Southern blot. FACS profiles and quantifications of Xs to Ys are shown to the right. The arrowheads shown in (A) and (E) represent replication forks stalling at sites other than RFB. doi:10.1371/journal.pgen.1005697.g003 Top2 and Sgs1-Top3 in rDNA Replication Termination  Top2 and Sgs1-Top3 in rDNA Replication Termination X-spike generating DNA structures have been observed in several studies both at the rDNA locus [39,40,41,42] and in other chromosomal regions [8,42]. To investigate if the X-spike observed in sgs1Δtop2 ts cells represents HR structures we investigated replication structures generated in the sgs1Δtop2 ts rad52Δ (Fig 5C) and sgs1Δtop2 ts rad51Δ (Fig 5D) triple mutants by 2D gel electrophoresis. In both mutants the X-spike was still present, and it persisted 100 minutes after α-factor release with a relative proportion of Xs to Ys similar to the one obtained in sgs1Δtop2 ts cells. The same was true for the X-spike formed in the BglIIA fragment covering most of the 35S transcription unit (S5 Fig). Thus, like checkpoint activation, X-spike formation in sgs1Δtop2 ts cells is not caused by unresolved recombination structures. This was further supported by the migration of the structures in 2D gels. Due to branch migration of junctions within HR structures these are expected to form X-spikes that extend with equal intensity over the entire spike, when experiments are performed in the absence of crosslinking agents, which is the case here [8]. In contrast, the X-structures in sgs1Δtop2 ts cells only gave rise to signals in the upper half of the spike and often with a punctuate nature of the spike (see e.g. Figs 4 and 5C). By the same token it is unlikely that the X-structures in sgs1Δtop2 ts cells represent hemicatenanes, which also form X-spikes in 2D gels [42].
Checkpoint activation is related to replication termination at RFB whereas X-spike formation is related to termination at other rDNA barriers Besides HR structures and hemicatenanes, forks converging during replication termination form X-structures. The X-spike in sgs1Δtop2 ts cells could therefore represent termination structures and be indicative of a failure during replication termination. If this is the case converging forks at rDNA replication fork barriers other than RFB should be responsible for the asymmetric X-structures constituting the major part of the X-spike (indicated by the stippled area in Fig  3B) and faulty termination at RFB should cause checkpoint activation. Barriers other than RFB have been demonstrated in the rDNA repeat both at ARS and the 5S transcription unit in the BglIIB fragment as well as in the 35S transcription unit in the BglIIA fragment [6]. Dots representing forks stalled at some of these positions were visible both in wt and sgs1Δtop2 ts cells (indicated by arrowheads in Fig 3A-3E). If asymmetric X-structures represent forks converging at these barriers and checkpoint activation is a result of faulty termination at RFB we speculated that a general weakening of all barriers would inhibit the accumulation of X-structures and diminish or abolish checkpoint activation. Rrm3 facilitates replication past replication fork barriers and has also been suggested to be involved directly in fork merging [8]. However, Pif1 has been demonstrated to counteract Rrm3 [6]. Therefore, if the DNA structures formed in sgs1Δtop2 ts cells would represent termination structures formed at different rDNA barriers we would expect that a deletion of PIF1 should reduce the formation of these structures either by directly facilitating fork merging at the barriers or by reducing fork stalling and thereby termination at these positions. To investigate this we deleted PIF1 and analyzed replication structures generated in the sgs1Δtop2 ts pif1Δ triple mutant by PFGE ( S6A Fig) and 2D gel electrophoresis (Fig 6A and S6B Fig) to see the implications of a PIF1 deletion for chr. XII migration and formation of asymmetric X-structures, respectively. Interestingly, chr. XII reentered the PFG after replication in the sgs1Δtop2 ts pif1Δ triple mutant and only wt levels of Xstructures were observed in 2D gels for the triple mutant in both BglII fragments, consistent with X-structures representing termination structures. An alternative explanation for the Pif1 dependency of the asymmetric X-structures could be that sgs1Δtop2 ts cells in the presence of Pif1 experience increased fork stalling at the different rDNA barriers, where the stalled forks are processed into X-structures in the mutant. However, we believe this is highly unlikely for several reasons. First, we do not observe increased stalling at RFB in sgs1Δtop2 ts cells as expected if the cells in general show increased stalling (Fig 3D). Furthermore, processing of stalled forks into X-structures is expected to require HR, which is not involved (Fig 5). Finally, we observed an increased Xs/Ys ratio in sgs1Δtop2 ts cells (Fig 3C), demonstrating an accumulation of X-structures rather than stalled forks, which indicates that processing of X-structures and not processing of Y-structures becomes the time limiting step in sgs1Δtop2 ts cells. Based on this, it seems unlikely that forks stall more often in sgs1Δtop2 ts cells than in wt cells. Rather the data suggest that when forks stalled at the different rDNA barriers are met by a fork coming Top2 and Sgs1-Top3 in rDNA Replication Termination from the opposite direction, fork merging becomes the time limiting step in sgs1Δtop2 ts cells, thus resulting in an accumulation of termination X-structures.
To investigate, if a PIF1 deletion affected checkpoint activation at RFB as expected if checkpoint activation is a result of faulty termination at this position, we investigated whether or not checkpoint activation occurred in sgs1Δtop2 ts pif1Δ cells. As seen in Fig 6B checkpoint activation was fully abolished in the triple mutant. Thus, a deletion of either PIF1 or FOB1 inhibits checkpoint activation at RFB, whereas only a deletion of PIF1 eliminates X-spike formation, consistent with a role of Pif1 at all rDNA replication fork barriers.
Taken together, the data suggest that lack of Top2 and Sgs1 becomes detrimental during replication termination at the rDNA locus in sgs1Δtop2 ts cells. Thus, replication termination at RFB causes checkpoint activation in these cells, which can only be abolished if either Fob1 is fully removed or the barrier is "loosened" as expected in the absence of Pif1. In contrast, termination at less strong rDNA barriers is merely delayed in sgs1Δtop2 ts cells and therefore accompanied by an accumulation of asymmetric termination X-structures, which decrease in amount over time and are sensitive to Top2/Top3 reactivation.
Neutral-Alkaline 2D gels reveal accumulation of termination structures at RFB and other stalling sites at the rDNA locus in the absence of Sgs1 and Top2 Neutral-Alkaline (N-A) 2D gels have earlier been used to verify the presence of unsolved termination structures [7]. With this method, X-shaped molecules generated due to termination at RFB are separated from replication forks stalled at RFB in the first dimension due to differences in their molecular weight. After migration in the second dimension, where denaturation will separate DNA strands, both termination structures at RFB and forks stalled at RFB will consist of full length template strands as well as nascent strands of approximately half the size ( Fig  7A). The template strands will thus form two dots located on the same horizontal line and the nascent strands will form two dots on a line below. Termination structures generated at positions other than RFB will form asymmetric X-structures. The template strands from these molecules will locate on the upper horizontal line, extending from the dot representing structures terminating at RFB towards the 2N dot. Besides full length parental strands, each asymmetric termination structure contains nascent strands of two sizes, which together make up the size of the parental strand. These strands will therefore in the second dimension form a "<" with legs emanating from the dot representing nascent strands from termination at RFB (Fig 7A, indicated by thick grey lines). Hemicatenanes and HR structures also form asymmetric Xs, but in contrast to termination structures both parental and nascent strands are full length and will locate on the upper horizontal line.
When DNA from sgs1Δtop2 ts cells was analyzed with this method, spots representing nascent strands from replication forks blocked at RFB (black arrowhead) as well as nascent strands from X-shaped structures representing forks converging at RFB (open arrowhead) were revealed (Fig 7B, second row). The emergence of these spots 40 minutes after α-factor release correlated with the appearance of corresponding spots in wt cells (Fig 7B, first row) in agreement with the observations from Neutral-Neutral 2D gels (Fig 3). However, whereas the spots representing forks converging at RFB had disappeared to background levels in wt cells 80 minutes after release, they remained at a higher level in sgs1Δtop2 ts cells (Fig 7C), although with a more diffuse appearance, strongly suggesting that termination at RFB was faulty. Furthermore, a smear extending as a "<" from the termination dot at RFB was present in wt and sgs1Δtop2 ts cells 60 minutes after release, which remained in the mutant but disappeared in wt (Fig 7D). The presence of DNA fragments causing the "<"-smear shows that termination  1N and 2N dots are indicated. (B) Genomic DNA was isolated from wt, sgs1Δtop2 ts , sgs1Δtop2 ts pif1Δ and sgs1Δtop2 ts fob1Δ cells at the indicated time points after release from α-factor, digested with BglII, and subjected to Neutral-Alkaline 2D gel analysis and structures are formed at positions other than RFB in the rDNA and they remain for a prolonged time in sgs1Δtop2 ts cells. In agreement with this, analysis of DNA isolated from the sgs1Δtop2 ts pif1Δ and sgs1Δtop2 ts fob1Δ triple mutants with this method demonstrated that the "<"-smear was absent in sgs1Δtop2 ts pif1Δ cells as expected, but still present in sgs1Δtop2 ts fob1Δ cells (except for the dots representing fork merging and stalling at RFB) (Fig 7B, third and fourth row and Fig 7D).

Discussion
In this paper we demonstrate that cells lacking Top2 and Sgs1-Top3 show two strong characteristics, checkpoint activation and X-spike formation. Both are connected to the rDNA locus, appear during late stages of replication, and are independent of HR. Interestingly checkpoint activation is Fob1-dependent, whereas X-spike formation is not. In contrast, structures responsible for X-spike formation are sensitive to Top2/Top3 reactivation, whereas those responsible for checkpoint activation are not. Thus, two different structures are formed in sgs1Δtop2 ts cells. This could either be due to a redundant function of Top2 and Sgs1-Top3 in two different processes or in a single process, having two different structural outcomes, when the enzymes are absent. Our results strongly suggest that it is the latter situation that is occurring, and that the process in which Top2 and Sgs1-Top3 are involved is replication termination. Thus, checkpoint activation occurs due to lack of Top2 and Sgs1-Top3 during replication termination at RFB, whereas X-spike formation is caused by a delay in replication termination at rDNA barriers other than RFB.
The results raise several questions. First, why do sgs1Δtop2 ts cells have problems during replication termination and why do these cause checkpoint activation at RFB and only a delay in termination at other fork barriers? Furthermore, why do sgs1Δtop2 ts cells show termination outside the normal RFB termination zone?
The finding that checkpoint activation occurs during termination at the strongest rDNA barrier whereas termination is merely delayed at other rDNA barriers when cells lack both Top2 and Sgs1-Top3, suggests that the problem experienced in the cells during termination is related to the nature of the barrier as well as to DNA topology. When a moving fork approaches a fork stalled at a barrier the topological tension between the forks increases and eventually leads to the formation of precatenanes behind the fork [13,14]. Several results have strongly suggested that the individual rDNA repeats are anchored to the nuclear membrane at RFB due to Fob1 interactions [43,44,45]. This fixation has been suggested to impose mobility constraints to the rDNA, and thus further increases the topological tension generated when forks converge at RFB. The structural consequence of the increased topological tension at RFB is speculative, but a checkpoint activating structure is finally formed, where either Top2 or Sgs1-Top3 can prevent formation of this structure as well as a deletion of either FOB1 or PIF1. In this topologically tense region we propose that Top2's role is to continuously decatenate precatenanes formed in the termination zone behind the replication forks. Top2-mediated decatenation will directly influence replication fork progression. However, the Fob1-and Pif1-dependency of checkpoint activation suggests that Top2 activity furthermore facilitates Rrm3-mediated Fob1 removal/fork merging as has been suggested earlier [8]. Our data demonstrate that Sgs1-Top3 work redundantly with Top2 in this process. In support of this, Rrm3 has Southern blotting using the P3 probe (Fig 2C). FACS profiles of samples taken throughout the experiments are shown to the right. (C) Quantification of forks converging at RFB at the indicated time points, where the signal at time point 0 is set to 1. Error bars represent STDEV from two to four independent experiments. (D) Quantification of forks converging outside RFB ("<"-smear) at the indicated time points, where the signal at time point 0 is set to 1. Error bars represent STDEV from two to four independent experiments. doi:10.1371/journal.pgen.1005697.g007 Top2 and Sgs1-Top3 in rDNA Replication Termination been demonstrated to be synthetic lethal with Sgs1 and Top3 [46,47]. An obvious role of Sgs1 and Top3 would be to unwind and decatenate, respectively, the DNA between the two converging forks. In support of this the E. coli homologs of Sgs1 and Top3 have been demonstrated to perform this reaction in vitro, when acting on a DNA substrate holding two closely located forks, thus mimicking a late replication structure [20].
Cozzarelli's lab has earlier demonstrated that the excess topological tension generated between replication forks promotes the formation of chickenfoot structures [48]. An equilibrium may thus exist between formation of precatenanes and chickenfoot structures. Another function of Sgs1-Top3 could therefore be to constantly revert chickenfoot structures to ensure replication fork progression and facilitate Rrm3-mediated Fob1 removal/fork merging together with Top2. In sgs1Δtop2 ts cells the equilibrium may be shifted towards the formation of chickenfoot structures. We observed that ssDNA was generated at the rDNA locus in a Fob1-dependent manner with the same timing as checkpoint activation. If chickenfoot structures are the cause of checkpoint activation they could be subject to DNA end resection, generating a ssDNA overhang, which recruits RPA and mediates checkpoint activation through Mec1. We would not be able to discern these resected strands in N-A 2D gels due to the smear produced by replication termination at rDNA barriers other than RFB. The two suggested roles for Sgs1/ Top3 are not mutually exclusive.
At the less strong barriers we expect that the topological tension generated when forks converge in the absence of Top2 and Sgs1-Top3 is allowed to slowly dissipate to more remote areas. This may be possible either because no anchorage is present to inhibit dissipation at these barriers or because the barriers are of a more transient nature. If the topological tension is lower than at RFB, chickenfoot structures may not be generated to an extent, where the amount of ssDNA exceeds the threshold required to trigger checkpoint activation. Rather, replication fork merging is merely delayed, causing an accumulation of asymmetric X-structures, which await dispersal of topological tension for final termination to take place. In correlation with this, the X-structures decreased in amount over time and were sensitive to late reactivation of Top2/Top3. Furthermore, X-structures were not visible in pif1Δ cells, indicating that the topological tension is easier to deal with when the barrier is more "loose". This observation furthermore supports that the function of Top2 and Sgs1-Top3 also at the less strong rDNA barriers is to facilitate Rrm3-mediated barrier removal/fork merging.
If asymmetric X-structures in sgs1Δtop2 ts cells represent termination structures, this means that termination to a great extent occurs outside the general RFB termination zone and thus that some replication forks pass RFB and are trapped at other barriers. Barriers other than RFB have been demonstrated in the rDNA including the 35S and 5S transcription units and the ARS element [6], where the nature of these barriers is unclear. Fork arrest was observed at some of these positions in wt as well as in the single and double mutants in the present study (Fig 3, arrowheads). Based on the migration of the asymmetric termination X-structures in 2D gels it seems as if mainly the transcription units act as fork barriers in sgs1Δtop2 ts cells besides RFB. At these positions the barrier effect may be caused by collision of the fork with the transcription apparatus or with topological tension generated in excess in these regions due to lack of Top2 [49]. Besides the demonstration that barriers other than RFB exist in the rDNA, it has also been demonstrated that not all leftward moving forks are arrested at RFB despite the general unidirectional replication mode at the rDNA locus [50,51]. Thus, relative to wt cells rrm3Δ cells show increased fork arrest both at RFB and at the other rDNA barriers, demonstrating that passage through RFB to some extent occurs in wt cells. RFB escape has also been demonstrated in pif1Δ cells, where the fraction of DNA in leftward moving forks was increased 2.5-fold relative to wt cells [6]. Very interestingly, when these observations are taken into account, the increased termination we see in sgs1Δtop2 ts cells at barriers other than RFB indicate that termination in general occurs outside RFB in wt cells, but only in sgs1Δtop2 ts cells is termination at these positions delayed, resulting in the accumulation of termination X-structures. In support of this, the observation that the relative level of X-structures to Y-structures was very high and remained high in sgs1Δtop2 ts cells suggests that termination rather than fork stalling becomes the rate limiting step in these cells. Furthermore, sgs1Δtop2 ts cells showed no sign of increased fork stalling.
Our data strongly suggests that Sgs1-Top3 becomes essential for replication termination when Top2 is absent, but that this redundant action of Top2 and Sgs1-Top3 is restricted to the rDNA locus. In sgs1Δtop2 ts fob1Δ cells, where the anchoring to the nuclear membrane of Fob1 bound rDNA repeats as well as unidirectional replication are abolished the situation is expected to mimic the situation outside the rDNA locus. However, under these conditions asymmetric termination X-structures were still observed in the rDNA, whereas we saw no accumulation of X-structures in sgs1Δtop2 ts cells, when analyzing replication structures at TER102 (S4 Fig) in correlation with earlier observations, where Top2, but not Top3, was found at termination sites outside the rDNA locus [8]. One explanation for this difference could be the high transcriptional activity at the rDNA locus. Transcription will increase the topological tension at the rDNA barriers which could create a need for Sgs1-Top3 besides Top2 and Rrm3 for efficient barrier dispersal/fork merging. Furthermore, it may well be that multiple copies are required to induce the robust cellular response observed in the rDNA. Thus, Top2 and Sgs1-Top3 may still play a role during termination outside the rDNA, but lack of Top2 and Sgs1-Top3 could cause less pronounced effects, which would be able to escape the detection limits of the assays employed in the present study.

Yeast strains
The employed strains were constructed using standard genetic techniques and are listed in S2 Table. All strains are derivatives of the original W303-1a.

Media and growth conditions
Unless otherwise stated, cells were grown to logarithmic phase in YPD media. Synchronization in G1 was achieved by transferring cells to YPD (pH 5.0) containing α-factor (2 μg/ml, Lipal Biochem) followed by incubation at 25°C for 150 min. Additional α-factor (1 μg/ml) was added after 1 hour of incubation to avoid escape from G1. To release the cells from arrest, they were washed once in water and transferred to fresh, pre-warmed (34°C) YPD medium. G2 arrest was achieved by adding nocodazole (Calbiochem) to a final concentration of 15 μg/ml.

Pulsed-field gel electrophoresis
Pulsed-field gel electrophoresis was performed as described in [52]. Cell cultures were grown to 3 x 10 7 cells/ml, and approximately 1.8 x 10 7 cells were cast into each plug to be run on the pulsed-field gel. The standard yeast genome size marker (Bio-Rad) was included on all gels. Gels were stained with ethidium bromide and transferred to Hybond XL membrane (GE Healthcare). Southern blotting was carried out using probes for chr. XII (Probe 1) and chr. II. Probes were amplified from purified yeast genomic DNA using 5'-CGCTTACCGAATTCTGCTTC and 5'-CTAGCATTCAAGGTCCCATT as forward and reverse primers, respectively, for chr. XII (Probe 1) and 5'-TCTCCGTCTTTAGTTGTTGC and 5'-GCCCTAGCAGTATTGCTTTG as forward and reverse primers, respectively, for chr. II. Experiments were performed 3-4 times with similar results.

FACS analysis
Samples were taken for FACS analysis during the various experiments and processed as described in [53]. Samples were analyzed in a BD FACSCalibur.
In situ kinase autophosphorylation (ISA) All steps of the ISA were as described in [26], except that 5 μCi/ml [γ-32 P] ATP was used. In short, protein extracts were generated from TCA-treated cells. For every sample, protein concentration was determined by Coomassie blue to allow loading of equal amounts of proteins on 10% SDS-polyacrylamide gels along with 5μl of a standard containing a known amount of MMS activated Rad53 ("+ control"). After gel electrophoresis proteins were transferred to PVDF filters (Immobilon-P, Millipore membranes). Filters were subjected to a denaturation/ renaturation protocol before the autophosphorylation reaction was performed by incubating membranes in kinase buffer in the presence of [γ-32 P] ATP. Dried filters were exposed on a Typhoon Trio+. After exposure, filters were re-probed with goat anti-Mcm2 (Santa Cruz) to check loading and allow comparison among different gels and mutants. Experiments were performed 2-3 times with similar results. MMS control ("+ control"): An Ay-120 culture (wt) with a density of 0.4 x 10 7 cells/ml was treated with 0.1% MMS for~60 minutes and harvested.

Two-dimensional gel electrophoresis
Yeast genomic DNA was isolated from 1 x 10 9 cells using Genomic-tip 20/G (QIAGEN) as described in [54]. After digestion with the BglII restriction enzyme (New England Biolabs) half of the purified DNA was subjected to Neutral-Neutral two-dimensional gel analysis as described in [55]. Southern blotting was carried out with the probes shown in Fig 2C, which were generated by PCR using genomic DNA as template. Probe 1 was generated as described above. For probe 2, recognizing the BglIIA fragment 5'-GTTTCTTTTCCTCCGCTT-3' and 5'-ATCTCTTGGTTCTCGCAT-3' were used as forward and reverse primers, respectively. For the probe near TER102 on chr. I 5'-GAAGGTTCAACATCAATTGATTGATTCTGCCGC CATGATC-3' and 5'-GCTTCCCTAGAACCTTCTTATGTTTTACATGCGCTGGGTA-3' were used as forward and reverse primers, respectively.
For Neutral-Alkaline two-dimensional gel electrophoresis BglII digested DNA was run on a Neutral gel in the first dimension. In the second dimension the excised DNA was run on a 1.5% agarose gel in 50 mM NaOH plus 1 mM EDTA at 4°C [7]. Probe 3 ( Fig 2C) used for southern blotting was made by PCR with 5'-CAGCCATAAGACCCCATC-3' and 5'-GCAGTTGGACGT GGGTTA-3' as forward and reverse primers, respectively, and genomic DNA as template.

Quantification of DNA structures
The intensity of DNA structures was measured using QuantityOne software. For Neutral-Neutral 2D gels the relationship between either X-structures and Y-structures or the RFB dot and Y-structures was calculated for each time point. Unless otherwise stated, the ratio at the different time points was related to the ratio at the 0 minute time point to allow comparison between strains. For the Neutral-Alkaline 2D gels the signal of the RFB dot or the "<"-smear at the different time points was related to the signal at the 0 minute time point. For the PFG's the ratio between chr. XII and chr. II re-entering the gel was calculated for each time point.

Fluorescent foci analysis
All strains harbored the pWJ1321 plasmid encoding Nop1-RFP and were therefore grown in synthetic complete media without histidine [56]. Cells were synchronized in G1 by treatment with α-factor for 150 minutes and released into SC-his medium supplemented with 100 μg/ml adenine at 37°C. Cell samples were collected, centrifuged at 2,000g and prepared for fluorescence microscopy as described in [57]. Fluorophores were visualized using band-pass CFP (31044) and RFP (41002c) filter sets from Chroma. Fluorescence images were acquired and processed using Volocity software (PerkinElmer). Statistical probabilities were calculated using Fisher's exact test (two-tailed). See S1 Table for morphology of cells included in the study.
Supporting Information S1 Fig. Fob1-dependent ssDNA-containing structures are formed in sgs1Δtop2 ts cells in late S/G2. wt, sgs1Δ, top2 ts and sgs1Δtop2 ts cells (A) or wt, fob1Δ, sgs1Δtop2 ts , and sgs1Δtop2 ts fob1Δ cells (B) having the endogenous Rfa1 protein (large subunit of RPA) tagged with CFP and Nop1-RFP expressed ectopically, were treated as in Fig 1A. At the indicated time points after release from α-factor, samples were withdrawn and fluorescent microscopy images were taken and analyzed.  Fig. sgs1Δtop2 ts cells do not show X-spike formation at a replication termination site outside the rDNA locus. Genomic DNA was isolated from wt and sgs1Δtop2 ts cells at the indicated time points after release from α-factor, digested with HindIII and subjected to 2D gel analysis and Southern blotting using a probe recognizing TER102 on chr. I [8]. FACS profiles are shown to the right. sgs1Δtop2 ts (B and C) or top2 ts top3 ts cells (D and E) were synchronized in G1 by α-factor at 25°C, released from α-factor into nocodazole (noc) at 34°C (37°C for top2 ts top3 ts ) and then kept at this temperature or transferred to 25°C either 25' or 60' after release from α-factor to reactivate Top2 (B and C) or Top2 and Top3 (D and E). Samples were withdrawn at the indicated time points and processed for ISA analysis of Rad53 (B and D) or 2D gel electrophoresis and Southern blotting with probe P1 (C and E). In  Fig 1A, except that cells were released at 34°C into nocodazole to block further cell cycling. Chromosomes were prepared from sgs1Δtop2 ts pif1Δ cells at the indicated time points after release from α-factor and visualized after pulsed-field gel electrophoresis by EtBr staining (upper panel) and by Southern blotting with a probe (P1) specific for chr. XII (lower panel). M, Chromosomal marker with indication of individual chromosomes to the left. (B) Genomic DNA was isolated from pif1Δ cells at the indicated time points after release from αfactor and subjected to 2D gel analysis and Southern blotting after digestion with BglII. P1, recognizing the BglIIB fragment was used as probe. FACS profiles are shown to the right. (EPS) S1 Table. Morphology of cells included in the fluorescence microscopy study. Cell morphology indicates cell cycle phase. Only cells that were synchronized in G1 with visible shmoo formation at 0' and progressed into budded cells after release into the S phase were counted. The increase in number of cells with foci coincides with the appearance of buds for all strains, indicating that foci are formed due to active replication. (DOCX) S2 Table. Strains used in this study. All strains are derivatives of W303-1a. (DOCX)