rsc mutants exhibit phenotypes characteristic of mutants defective in chromosome segregation. (A) sfh1-1ts activates the MAD1-dependent spindle checkpoint pathway. Upper panel: Flow cytometric analysis of DNA contents in SFH1 (BLY46), mad1 (BLY295), sfh1-1ts (BLY75-1), and sfh1-1ts mad1 (BLY296) strains at the times indicated. Cells grown to mid-logarithmic phase at 25°C were shifted to 37°C, and aliquots were removed for analysis at the times indicated. The number of cells was plotted versus the relative intensity of emitted light. Lower panel: At the times indicated, the SFH1 (open circles), mad1 (open triangles), sfh1-1ts (open squares), and sfh1-1ts mad1 (solid circles) cells described above were removed from 37°C and plated onto YPD medium at 25°C. Viability was determined as the number of cells capable of forming colonies at 25°C. Values reported are the averages for two independent determinations with <15% error. (B) Homozygous sth1-3ts diploid cells exhibit increased 2:0 missegregation of a chromosome fragment. STH1/STH1 (BLY363) and sth1-3ts/sth1-3ts (BLY394) cells carrying a single copy of CFIII were incubated at 37°C for 4 h before shifting back to 25°C. Red sectors indicate loss of chromosomes, and white sectors indicate gain of chromosomes. Half-sectored colonies were scored as R/W (half red/half white) or R/P (half red/half pink) after 2 to 4 days of incubation. The rates of CFIII nondisjunction (2:0 segregation) and loss (1:0 segregation) were calculated by dividing the number of half red/half white and half red/half pink cells, respectively, by the total number of pink parental cells indicated. The data from two independent experiments were combined.

sth1-3ts and sfh1-1ts mutations interact with a histone H4 mutation, hhf1-20. (A) sth1-3ts suppresses the temperature-sensitive phenotype of hhf1-20 mutants. Growth of sth1-3ts hhf1-20 double mutants (BLY546) was compared to that of hhf1-20 (BLY415), sth1-3ts (BLY49), and wild-type (WT, BLY76) cells at 25°C and 34°C. Plates were incubated for 2 to 3 days. Four independently isolated double mutant strains were tested. (B) The sfh1-1ts mutation combined with hhf1-20 results in synthetic growth defects. The growth of wild-type (BLY278), sfh1-1ts (BLY353), hhf1-20 (BLY415), and sfh1-1ts hhf1-20 (BLY552) cells on YPD was compared at the indicated temperatures after 3 days.

Rsc8p associates with centromeric and flanking chromosomal regions in vivo. (A) Rsc8p is localized to centromeric and flanking regions. Chromatin prepared from extracts of cells expressing Mcd1p-HA (BLY397) or Rsc8p-HA (BLY298), and untagged Mcd1p (BLY398) or Rsc8p (BLY301) grown to mid-logarithmic phase in YPD was immunoprecipitated (IP) with anti-HA antibodies. Coimmunoprecipitated DNAs were amplified by PCR with primers specific for the indicated chromosomal loci. (B) Rsc8p localizes to CEN3-flanking and CEN1 regions in S- and G₂/M-phase-synchronized cells. Chromatin was prepared from cells expressing HA-tagged Rsc8p (BLY298) synchronized in S or G₂/M phase by treatment with hydroxyurea or nocodazole, respectively, for 3 h at 25°C. (C) Association of Rsc8p with CEN DNA and flanking regions is defective in sth1-3ts mutants. Chromatin was prepared from mid-logarithmic-phase RSC8-HA (BLY298), RSC8 (BLY301), and sth1-3ts RSC8-HA (BLY549) cells grown at 25°C or 37°C for 8 h. A schematic map showing the positions of the PCR products relative to CEN3 is shown.

Mif2p and Cse4p remain associated with centromeres in sth1-3ts mutant cells. Chromatin was prepared from extracts of wild-type STH1 or mutant sth1-3ts cells expressing HA-tagged Cse4p (PM1311 and PM1312, respectively). Cells grown to mid-logarithmic phase in YPD medium at 23°C were first incubated with α-factor for 3 h to arrest cells in G₁ phase (≈85%). Half of each culture was then shifted to 37°C in the continued presence of α-factor for 30 min, after which cells were washed with YPD containing 0.1 mg of pronase per ml at 23°C or 37°C and released into fresh YPD medium containing 0.1 mg of pronase per ml and nocodazole. Cells were incubated for an additional 3.5 h before processing for chromatin immunoprecipitation. Duplicate stained CEN3 PCR products (except for lane 7, upper panel) were imaged and quantified. No, mock immunoprecipitation without antibody.

RSC interacts genetically with components of the kinetochore. (A) sth1-3ts interacts with mutations in kinetochore components. The genotypes refer to the following strains: wild type (WT, BLY76), sth1-3 (BLY49), ctf14-42 (BLY503), sth1-3 ctf14-42 (BLY528), cse4-1 (BLY500), sth1-3 cse4-1 (BLY536), mif2-3 (BLY416), sth1-3 mif2-3 (BLY531), ndc10-1 (BLY506), and sth1-3 ndc10-1 (BLY532). Strains were grown on YPD plates at the indicated temperatures for 3 to 4 days. Four independently isolated double mutant strains were tested on each plate. (B) sth1-3ts mutations in combination with either cse4-1 or mif2-3 mutations cause enhanced hypersensitivity to TBZ. Tenfold serial dilutions of mid-logarithmic-phase cells were spotted onto YPD plates containing 0 or 60 μg of TBZ per ml. Growth of wild-type (BLY76), sth1-3ts (BLY49), cse4-1 (BLY500), sth1-3ts cse4-1 (BLY536), mif2-3 (BLY416), and sth1-3ts mif2-3 (BLY531) cells was compared at 25°C after 3 to 4 days. (C) The sfh1-1ts mutation interacts synthetically with the ndc10-1 mutation. Wild-type (BLY278), sfh1-1ts (BLY353), ndc10-1 (BLY506), and sfh1-1 ndc10-1 (BLY538) strains were grown on YPD plates at the temperatures indicated for 3 to 4 days. (D) Increased dosage of CSE4 partially suppresses the temperature sensitivity and TBZ sensitivity of sfh1-1ts mutants. Equal numbers of tenfold serially diluted mid-logarithmic-phase sfh1-1ts cells (BLY353) carrying YEp24 or YEp24-derived high-copy plasmids expressing SFH1 (pYC5H), CSE4 (pPY3), or NDC10 (pPM47) were spotted onto SC plates lacking uracil and incubated at 25°C or 31°C or onto SC plates lacking uracil plates containing 60 μg of TBZ per ml and incubated at 25°C. Cell growth was compared after 3 to 4 days. (E) sth1-3ts mutations cause synergistic chromosome instability when combined with a CDEI but not a CDEII mutation. The stabilities of chromosome fragments containing either a wild-type CEN or mutant CEN sequence CDEI(8-C) or CDEII(+45) in STH1 (BLY361, BLY429, and BLY424, respectively) and sth1-3ts (BLY360, BLY565, and BLY566, respectively) cells were determined by plating cells onto adenine indicator plates. Cells were grown at 34°C and photographed after 3 to 4 days. Red sectors indicate missegregation of the chromosome fragment.

Sth1p interacts physically with the histone H3 variant Cse4p and histones H3, H4, and H2B. (A) Sth1p interacts with Cse4p in the yeast two-hybrid assay. β-Galactosidase activity was assayed in the lexAop-lacZ (BLY2) reporter strain expressing pLexA or pLexA₂₀₂-Sth1_1-1359 and Gal4p activation domain (GAD) or GAD fusion proteins expressed from pACT2, pJL484, pJL486, pJO196, pGAD-CTF19, or pIT331. β-Galactosidase activity in permeabilized cells was determined as described previously (11). No significant interactions were detected in cells expressing the LexA DNA-binding partner alone and GAD fusion proteins or in cells expressing the GAD activating partner alone and LexA₂₀₂-Sth1_1-1359. The interaction between LexA₂₀₂-Sth1_1-1359 and GAD-Cse4p was confirmed in β-galactosidase liquid assays (data not shown). AD, activation domain. (B) Sth1p interacts with Flag-tagged histones H2B, H3, and H4 in vivo. Anti-Flag M2 monoclonal antibody affinity resin was incubated with whole-cell extracts (WCE) prepared from cells carrying plasmids expressing Flag-H2B (BLY309), H2B (BLY283), Flag-H3 (BLY573), or Flag-H4 (BLY575) and cells carrying pRS315 (BLY572) and pRS425 (BLY574) as controls for Flag-H3 and Flag-H4, respectively. Immune complexes were separated on SDS-4 to 20% polyacrylamide gradient or SDS-15% acrylamide gels and immunoblotted with anti-Sth1p (1:1,000) polyclonal or anti-Flag M2 (1:300) monoclonal antibodies. Lanes 1 and 2, 1/83 of input lysate for both anti-Sth1p and anti-Flag immunoblots; lanes 3, 4, 5, and 6, entire immunoprecipitation pellets for both anti-Sth1p and anti-Flag Western blots. Immunoprecipitations (IPs) were performed in the presence of 250 U of DNase I except for lanes 5 and 6, from which DNase I was omitted. Nonspecific proteins comigrated with immunoprecipitated Flag-H3 and Flag-H4 proteins (compare lanes 3 and 4). (C) Sth1p interacts directly with histones H3, H4, and H2B by Far-Western analysis. Samples of acid-soluble enriched histones were separated by SDS-21% PAGE, blotted to polyvinylidene difluoride membranes, and probed with radiolabeled Sth1p (Sth1p). A parallel lane of transferred histones was stained with Ponceau S (Stain). H3* is a spontaneous breakdown product of histone H3. (D) Sth1p interacts directly with the N termini of histones H3 and H4. [³⁵S]methionine-labeled Sth1p was incubated with GST (lanes 2) or GST fusion proteins containing the N termini of histone H3 (amino acids 1 to 46) or H4 (amino acids 1 to 34) (lanes 3), H3 (1 to 25) or H4 (1 to 16) (lanes 4), or H3 (21 to 46) or H4 (15 to 34) (lanes 5) (28) immobilized on glutathione-Sepharose beads. We analyzed 25% of the input fractions and 100% of the bound and unbound fractions by SDS-8% PAGE, and labeled Sth1p was visualized by PhosphorImager analysis. Similar results were observed in at least four independent experiments.

Centromeric and flanking chromatin structure is altered in rsc mutants. (A) DraI accessibility to CDEII of CEN3 is increased in sth1-3ts mutants. Nuclei isolated from sth1-3ts (BLY49) grown at 25°C or hhf1-20 (BLY413), sth1-3ts (BLY49), and wild-type (WT, BLY76) cells grown at 35°C for 8 h were first digested with DraI. Southern blot analysis of the purified DNA subsequently digested with HindIII is shown. M, ≈1.4-kb DraI-HindIII genomic fragment as a marker. The 7.0-kb band is the intact HindIII fragment uncut by DraI. The fraction of CDEII chromatin digested by DraI is shown as a percentage. (B) Pericentromeric chromatin structure is altered in the sfh1-1ts mutant. Indirect end labeling of microccocal nuclease-digested chromatin prepared from SFH1 (BLY278) and sfh1-1ts (BLY353) cells grown at 25°C or 37°C for 8 h. CDEI-proximal pericentromeric chromatin structure is shown. The arrowheads indicate regions in the mutant with altered digestion patterns; solid circles indicate the corresponding regions in wild-type cells. N, naked DNA control.

rsc mutants missegregate authentic chromosomes. (A) Sister chromatid segregation is defective in sth1-3ts mutants. STH1 (BLY520) and sth1-3ts (BLY526) cells marked at CEN1 were grown to mid-logarithmic phase at 25°C, diluted, and shifted to 37°C for 8 h. Representative mutant cells with separated sister chromatids in one cell body are shown (columns b to d). Mutant sth1-3ts cells (BLY493) marked at URA3 (35 kb from CEN5) with separated sister chromatids are shown in column e. Nuclei were visualized by 4,6-diamidino-2-phenylindole (DAPI) staining. Nom, Nomarski images. (B) Spindle morphology is aberrant in sth1-3ts mutants. Mid-logarithmic-phase wild-type (BLY570) and sth1-3ts (BLY568) cells expressing the GFP-Tub1p fusion protein were grown at 37°C for 8 h. Microtubule structures were analyzed by fluorescence microscopy. (C) Sister chromatid segregation is delayed in sth1-3ts cells. Wild-type (BLY520) and sth1-3ts (BLY526) strains marked at CEN1 were arrested in G₁ with α-factor, released at 37°C, and scored for sister chromatid separation and segregation. The percentages of cells with segregated, separated, and unseparated sister chromatids 100 min after α-factor release are plotted.