Figure 1.
Schematic of DNA supercoiling, SCIs and topoisomerases.
(A) Schematic figure of changes in DNA topology at the replication fork. Progressive separation of the parental DNA strands by the replication machinery leads to the accumulation of positive supercoils ahead of the replication fork. These can be resolved by both Top1 and Top2 topoisomerases in budding yeast. Sister chromatid intertwinings (SCIs) can be formed through rotation of the fork. Resolution of the SCIs by Top2 ensures full chromosome segregation during mitosis. (B) Schematic figure of changes in DNA topology due to transcription, positive supercoils accumulate ahead of the transcription machinery, while negative ones are found behind. (C) Top2 acts on SCIs, while both Top1 and Top2 can resolve supercoils.
Figure 2.
The chromosomal association of Smc6 depends on cohesin.
(A) Comparison of ChIP-on-chip and ChIP-seq. Chromosomal localization of Smc6-FLAG as determined by ChIP-on-chip (upper panel) and ChIP-seq (middle panel). The lowest panel shows a ChIP-seq map from a control experiment performed on cells lacking FLAG-tagged proteins. (B) Chromosomal association of Smc6-FLAG in G2/M-arrested scc1-73 cells as determined by ChIP-seq. In both (A) and (B), cells were arrested in G2/M after a synchronous S-phase at 35°C before sample preparation. The asterisk (*) denotes a false positive peak. The Y-axis shows fold enrichment of ChIP/whole cell extract (WCE) in log scale for ChIP-on-chip and in linear scale for ChIP-seq, while the X-axis shows chromosomal positions. Blue horizontal bars in the panel describing the genomic region denote open reading frames, while red and green vertical lines denote replication origins (ARS) and centromeres (CEN), respectively. (C) Chromosomal association of Smc6-FLAG at selected chromosomal positions in indicated strains as determined by ChIP-qPCR. Cells were arrested in G2/M after a synchronous S-phase at 35°C, or after G1 arrest (G1), before sample preparation. Chromosome number and distance from the left telomere of each analyzed position is indicated below the corresponding group of bars. Y-axis displays the amount of DNA in ChIP fraction in relation to input. Each bar represents the mean of three independent experiments with standard deviations indicated. Statistical analysis was performed using the Student's two-tailed t-test. P-values are illustrated in the figures as follows: n. s. = not significant (p>0.05); * = significant (p<0.05); ** = significant (p<0.01); *** = significant (p<0.001). Maps displaying Smc6 enrichment at the corresponding sites as determined by ChIP-seq are displayed in Figure S2. (D) Chromosomal association of Smc6-HA as determined by immunofluorescence of chromosome spreads. Indicated SMC6-3HA-expressing cells were arrested in G2/M after a synchronous S-phase at 35°C. Samples were collected and chromosome spreads were prepared and stained to detect the HA-epitope tag on Smc6. Box whisker plots representing the signal intensity in arbitrary units (AU) from quantifications of at least 50 cells for each sample are shown. Statistical analysis was done using a two-tailed t-test with Welch's correction. P-values are as follows: Wild-type (no tag) vs. SMC6-3HA, p<0.0001 (****); SMC6-3HA vs. scc1-73 SMC6-3HA, p<0.0001 (****); SMC6-3HA vs. top2-4 SMC6-3HA, p = 0.0006 (***);scc1-73 SMC6-3HA vs. top2-4 scc1-73 SMC6-3HA, p = 0.0002 (***); top2-4 SMC6-3HA vs. top2-4 scc1-73 SMC6-3HA, p<0.0001 (****).
Figure 3.
The chromosomal association of Smc5/6 depends on sister chromatid cohesion.
(A–C) Chromosomal association of (A and C) Smc6-FLAG and (B) Scc1-FLAG in indicated strains as determined by (A and B) ChIP-seq and (C) ChIP-qPCR. All cells were arrested in G2/M after a synchronous S-phase at 35°C before sample preparation. Note that the false positive signal in denoted (*) is mostly absent in SCC1-FLAG cells. Figure details and statistical analysis are described in Figure 2. In (C), results for Smc6-FLAG in wild-type cells are identical to those displayed in Figure 2C, and shown for comparison.
Figure 4.
Smc5/6 is enriched on replicated chromosomes in between convergently transcribed genes close to centromeres, and the level of enrichment increases if the centromeres are distant from a chromosome end.
(A–C) Chromosomal localization of Smc6-FLAG during G1 (A) and S-phase (C), or bromodeoxyuridine (BrdU) incorporation (B), as determined by ChIP-seq. In (B and C), cells were arrested in G1 and subsequently released into media containing BrdU in the presence of hydroxyurea (HU), or at 18°C as indicated. Samples were collected in HU and 1 and 2 hours after the release. ChIP-seq analysis was performed using anti-FLAG (A and C), or anti-BrdU antibodies (B). (D) Chromosomal localization of Smc6-FLAG, Nse4-FLAG and Scc1-FLAG in G2/M-arrested cells. Samples were collected in a nocodazole-induced G2/M-arrest after a synchronous S-phase at 35°C. (E) Chromosomal localization of Smc6-FLAG cells arrested in telophase due to non-functional Cdc15 mitotic exit network kinase. Samples were prepared as in (D) but released from G1-arrest in nocodazole-free medium. Panel details as in Figure 2 with the addition of vertical dashed lines showing that Smc6-FLAG and Nse4-FLAG co-localize with Scc1 in between convergently oriented genes in the G2/M-arrest. (F–H) Scatterplots comparing Smc6-FLAG enrichment with full chromosome length (F), chromosome arm length (G), or the distance from the centromere to the nearest telomere (i. e. the length of the shorter chromosome arm) (H). Enrichment values represent Smc6-FLAG enrichment in 100 kb regions spanning the centromeres (F and H) or in 50 kb regions to the left or right of the centromere (G). Enrichment values from ChIP-seq performed on a control strain lacking tagged proteins were subtracted for each region (see Figure S4). Correlation values are indicated in each graph. Chromosome 12 was excluded from the scatterplots due to the unknown length of the rDNA present on that chromosome.
Figure 5.
Smc5/6 accumulates on chromosomes after Top2 inhibition, and co-localizes with cohesin in both wild-type and top2-4 cells.
(A–D) Chromosomal association of (A, C and D) Smc6-FLAG and (B) Scc1-FLAG in indicated strains as determined by (A–C) ChIP-seq and (D) ChIP-qPCR. All cells were arrested in G2/M after a synchronous S-phase at 35°C before sample preparation. The ChIP-seq maps displays a chromosomal region spanning 100–200 kb from the left telomere of chromosome 7. Panel details and statistical analysis are described in Figure 2. In (D), results for Smc6-FLAG in wild-type cells are identical to those displayed in Figure 2C, and shown for comparison. (E) Overlap of Smc6 and Scc1 binding sites on chromosome arms. For annotation of Smc6 and Scc1 binding sites, see Material and Methods.
Figure 6.
The increase of chromosome-bound Smc5/6 in top2-4 cells requires passage through S-phase at restrictive temperature.
(A) Chromosome arm association of Smc6-FLAG in G2/M-arrested top2-4 cells as determined by ChIP-seq. Samples were collected after 1 hour temperature increase to 35°C under maintained arrest. Note the lack of accumulation of Smc6-FLAG in this region, as compared if Top2 is inactivated during replication (Figure 5A). (B) Analysis of Smc6-FLAG in G1-arrested wild-type and top2-4 cells by ChIP-on-chip. Samples were collected after a 30 minutes at 35°C in a sustained G1-arrest. A region spanning 100–200 kb from the left telomere of chromosome 7 is shown. Panel details are described in Figure 2.
Figure 7.
The chromosomal association of Smc6 does not depend on DSB formation or recombination.
(A and B) Chromosome arm association of Smc6-FLAG in G2/M-arrested mre11Δ, top2-4 mre11Δ, rad52Δ, top2-4 rad52Δ cells as indicated, determined by ChIP-seq (A) and ChIP-qPCR (B). Samples were collected in G2/M after a synchronous S-phase at 35°C, nonpermissive for the top2-4 allele. In (A), a region spanning 100–200 kb from the left telomere of chromosome 7 is shown. Panel details for (A) and (B) are described in the legend of Figure 2. (C) Western blot of Rad53 and actin in wild-type and top2-4 cells arrested in S-phase by HU or in G2/M by nocodazole, as indicated.
Figure 8.
The chromosomal association of Smc6 does not depend on replication fork stalling.
(A) ChIP-seq analysis of FLAG-tagged DNA polymerase II subunit Dpb3 in G2/M-arrested wild-type and top2-4 cells. Compare to the enrichment of Smc6-FLAG seen at specific loci in this region in Figure 5A (B) ChIP-on-chip analysis of chromosome association of FLAG-tagged Dpb3 (upper panel) and BrdU-incorporation (lower panel) in HU-arrested S-phase cells. (C) Chromosome arm association of Smc6-FLAG in G2/M-arrested rrm3Δ cells. G2/M-samples were collected after a synchronous S-phase at 35°C, nonpermissive for the top2-4 allele (A). For analysis in S-phase, cells were arrested in G1 and subsequently released into media containing BrdU and HU. Samples were collected 1 hour after the release (B). Analysis was performed using anti-FLAG (A, upper panel in B, and C), or anti-BrdU antibodies (lower panel in B). A region spanning 100–200 kb from the left telomere of chromosome 7 is shown, panel details are described in Figure 2.
Figure 9.
Chromosomal regions where Smc5/6 accumulates after Top2 inhibition show no sign of persistent replication or recombination intermediates.
(A) Chromosomal localization of Smc6-FLAG as determined by ChIP-seq at two loci, UBP10-MRPL19 and MPP10-YJR003C, showing abundant Smc6-FLAG binding in top2-4. The lowest panel shows a ChIP-seq map from a control experiment performed on cells lacking FLAG-tagged proteins. Panel details and cellular growth conditions are as described in the legend of Figure 2. In the top panel describing genomic features, arrows and chromosomal positions denote the restriction sites for PstI, used to produce the analyzed fragments. (B) Two-dimensional gel electrophoresis of UBP10-MRPL19 (left) and MPP10-YJR003C (right) in wild-type and top2-4 cells. Cell cycle progression monitored by fluorescence-activated cell sorting (FACS) is shown below and time-points of sample preparation are indicated. Membranes were first probed against UBP10-MRPL19 (left), then stripped and re-probed against MPP10-YJR003C (right), leaving some residual signal from UBP10-MRPL19 in the MPP10-YJR003C blots (white arrows). (C) Two-dimensional gel electrophoresis after DNA isolation using CTAB-extraction to preserve X-shaped molecules, of ARS305 (left) and UBP10-MRPL19 (right) in wild-type and top2-4 cells. The ARS305 containing fragment was produced by digestion using EcoRI and HindIII, and is a positive control for X-shaped molecule isolation (white arrowheads). Cell cycle progression monitored by FACS is shown below and indicates the time-points of sample preparation.
Figure 10.
Restoration of Top2 function after replication removes Smc5/6 from chromosomes.
(A) Segregation of a fluorescently labeled region located 72 kb from the right telomere of chromosome 5 in wild-type and top2-4 cells. After G1-arrest at permissive temperature for top2-4 (23°C), cells were incubated at the restrictive temperature 35°C during 30 minutes before release at the high temperature. Segregation was scored in next G1 without any intervening arrest, or after 1 hour in G2/M at 23°C or 35°C, as indicated. Missegregation was defined as events when chromosome 5 was absent from mother or daughter cell 70 minutes after the start of imaging, and only scored in cells in which spindle elongation had occurred. (B) Experimental setup in C–E. (C) Southern blot analysis of the plasmid pRS316, isolated from top2-4 cells at time points indicated in (B). pRS316 is in supercoiled and relaxed monomeric forms in G1 (lane 1 and 5, compare to control plasmid isolated from bacteria, lane 9). After an S-phase at 35°C, pRS316 accumulates as a high molecular weight form (lane 2). This is supercoiled dimeric plasmids, as shown by its transformation into a ladder of relaxed dimeric molecules after treatment with the nicking enzyme Nb.BtsI (lane 10 and 11). Reactivation of Top2 through temperature down-shift resolves dimers into supercoiled and relaxed monomeric plasmids (lane 3), while maintained inactivation leaves the dimers unresolved (lane 4). After replication at 23°C, the plasmids remain in the monomeric forms, also after top2-4 inactivation in G2/M (lanes 5–8). (D) Chromosomal association of Smc6-FLAG at selected chromosomal positions as determined by ChIP-qPCR. Chromosome number and distance from the left telomere of each position is indicated below the corresponding bar. Restoration of Top2 function by temperature down-shift in G2/M leads to dissociation of Smc6 from all tested binding sites except at the core centromere of chromosome 9. Results for Smc6-FLAG in top2-4 cells are identical to those displayed in Figure 5D, and shown for comparison. (E) Association of Smc6-FLAG to a region spanning 100–200 kb from the left telomere of chromosome 7 as determined by ChIP-seq. top2-4 cells were taken through a synchronous S-phase at 35°C, arrested in G2/M, and samples were prepared 1 hour after a temperature down-shift to 23°C (upper panel), or after 1 hour at maintained, high temperature (lower panel).
Figure 11.
Chromosome segregation in wild-type, top2-4, scc1-73 and top2-4 scc1-73 cells.
(A) Representation of experimental setup. Wild-type, top2-4, scc1-73, or top2-4 scc1-73 cells harboring multiple copies of tetracycline operators at a specific chromosomal region, and expressing GFP-tagged tubulin and tdTomato-marked tetracycline repressors, were imaged during progression through one synchronous cell cycle under conditions which inactivated both mutant alleles. Elongation of the tubulin spindle was used as time point zero (0). Chromatid separation was defined as the moment when the tdTomato signal was split into two, segregation when each of the chromatid-marking dots was found in mother and bud cell, respectively. On the X-axis, bars at time point 0 represent the sum of separation events in the images collected at 0 and 0.5 minutes, time point 1 minute the sum of separation events in the frames of 1 and 1.5 minutes etc. Cells in which chromatids do not separate during the 70 minutes of imaging fall in into the category of events which are marked with an asterisk on the X-axis. If segregation occurs within 5 minutes of separation, the cells fall into the “green-bar-category” on the Z-axis (≤5 minutes). If segregation occurs more than 5 minutes after separation, the cells fall into the “red-bar-category” on the Z-axis (>5 minutes). Cells that do not segregate their chromatids during the entire 70 minutes of imaging are placed into the “black-bar-category” on the Z-axis (triangle). (B-D) Separation and segregation of chromosomes 4 (B), 1 (C), 5 (D) in wild-type and top2-4 cells. (E and F) Separation and segregation of chromosomes 1 (E) and 5 (F) in scc1-73 and top2-4 scc1-73 cells. The tetracycline operators are integrated 35 kb from the centromeres on chromosomes 1, 5 and 4. This places these markers at 44, 117 and 1045 kb away from the telomeres. On chromosomes 5 and 4 the telomere proximal sites are placed 350 and 995 kb away from centromere, respectively. This places them at 72 and 85 kb away from respective telomeres.
Figure 12.
Chromosome-bound Smc5/6 predicts the degree of missegregation after Top2 inactivation.
(A–C) Segregation of a fluorescently labeled region located ∼995 kb from the centromere of chromosome 4 (A), and ∼350 kb from centromere of chromosome 4 (B), and 5 (C), in wild-type and top2-4 cells. The level of Smc6 enrichment in the 350 kb region between the centromere and the tetracycline operators on chromosome 4 and 5, calculated as in Figure 4, is indicated in the schematic maps of the chromosomes above each panel. Cells were first arrested in G1 at permissive temperature for top2-4 (23°C), and thereafter either incubated at the restrictive temperature 35°C during 30 minutes before release at 35°C, or released into a G2/M-arrest at 23°C. In the G2/M-arrest the temperature was raised to 35°C for 1 hour before release at the high temperature. Segregation was subsequently scored as in (Figure 11A) in both cell populations.
Figure 13.
Smc5/6 promotes segregation of intertwined chromatids without perturbing cohesin removal.
(A) Segregation of a fluorescently labeled region located 35 kb away from the centromere of chromosome 1 in indicated strains. Cells were first arrested in G1 at 23°C, and then incubated at 35°C for 30 minutes before release at maintained temperature. Segregation was scored during following anaphase as in Figure 11A. (B) FACS analysis of cdc15-2 SCC1-FLAG and cdc15-2 smc6-56 top2-4 SCC1-FLAG cells. Cells were first arrested in G1 at 23°C, and then incubated at 35°C for 30 minutes before release at maintained temperature in the absence or presence of nocodazole. Samples for analysis of nuclear division, Scc1 proteins levels in solution and on chromatin were collected 2 hours after release. (C) Nuclear division 2 hours after release. Cells were fixed in 3,7% formaldehyde for 10 minutes and stained with DAPI and analyzed under microscope. Cells were scored into either of the following three categories; one DNA mass, one stretched DNA mass bridging the mother cell and daughter bud or two separated DNA masses, one in the mother cell and one in the daughter bud. Cells released in the presence of nocodazole showed no sign of nuclear division. In contrast, cdc15-2 SCC1-FLAG cells released in the absence of nocodazole had undergone anaphase and were arrested in telophase with two DNA masses. cdc15-2 smc6-56 top2-4 SCC1-FLAG cells released in the absence of nocodazole had commenced anaphase but failed to separate the DNA into two masses, displaying one stretched DNA mass. (D) Scc1 protein levels detected by western blot using an anti-FLAG antibody. Actin was used as loading control. (E) Scc1-FLAG chromosome association as determined by ChIP-qPCR. Note that both total levels and chromosome associated Scc1 are similarly reduced in the two strains when arrested in a cdc15-2-induced telophase arrest (without nocodazole).
Figure 14.
Model describing the connection between chromosome segregation and the chromosomal association of Smc5/6 and cohesin.
Summary of how the chromosomal association of Smc5/6 in metaphase (left) correlates with chromosome segregation in anaphase (right) in wild-type and top2-4 cells, in which top2-4 is inactivated either prior or after DNA replication. See discussion for detailed description.