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. 2000 Oct 30;151(3):613-26.
doi: 10.1083/jcb.151.3.613.

Pds5p is an essential chromosomal protein required for both sister chromatid cohesion and condensation in Saccharomyces cerevisiae

T Hartman  1 , K Stead, D Koshland, V Guacci
Affiliations

Pds5p is an essential chromosomal protein required for both sister chromatid cohesion and condensation in Saccharomyces cerevisiae

T Hartman et al. J Cell Biol. .

Abstract

The PDS5 gene (precocious dissociation of sisters) was identified in a genetic screen designed to identify genes important for chromosome structure. PDS5 is an essential gene and homologues are found from yeast to humans. Pds5p function is important for viability from S phase through mitosis and localizes to chromosomes during this cell cycle window, which encompasses the times when sister chromatid cohesion exists. Pds5p is required to maintain cohesion at centromere proximal and distal sequences. These properties are identical to those of the four cohesion complex members Mcd1p/Scc1p, Smc1p, Smc3p, and Scc3p/Irr1p (Guacci, V., D. Koshland, and A. Strunnikov. 1997. Cell. 91:47-57; Michaelis, C., R. Ciosk, and K. Nasmyth. 1997. Cell. 91:35-45; Toth, A., R. Ciosk, F. Uhlmann, M. Galova, A. Schleiffer, and K. Nasmyth. 1999. Genes Dev. 13:307-319). Pds5p binds to centromeric and arm sequences bound by Mcd1p. Furthermore, Pds5p localization to chromosomes is dependent on Mcd1p. Thus, Pds5p, like the cohesin complex members, is a component of the molecular glue that mediates sister chromatid cohesion. However, Mcd1p localization to chromosomes is independent of Pds5p, which may reflect differences in their roles in cohesion. Finally, Pds5p is required for condensation as well as cohesion, which confirms the link between these processes revealed through analysis of Mcd1p (Guacci, V., D. Koshland, and A. Strunnikov. 1997. Cell. 91:47-57). Therefore, the link between cohesion and condensation is a general property of yeast chromosomes.

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Figures

Figure 1
Figure 1
Characterization of pds5 mutants. (A) Ts− phenotype of pds5 cells. Wild-type (VG982-6A), pds5-1 (VG986-5B), pds5-2 (VG987-5C), and pds5-3 (VG988-1C) cells grown at 23°C in YPD liquid were plated in 10-fold serial dilutions on YPD and incubated at 23°, 30°, and 37°C. (B) Cell cycle–dependent lethality of pds5 cells. Strains in A were arrested at 23°C in either G1, S, or mid-M phase, incubated at 37°C while arrested, and then plated on YPD at 23°C to determine percent viability (Materials and Methods). Data from three independent experiments was used to generate error bars. (C) Cloning of the PDS5 gene. Strains in A were transformed with either a CEN vector alone or one bearing the PDS5 ORF.
Figure 2
Figure 2
Cell cycle progression in wild-type and pds5 mutant cells. (A) DNA content of cells. Wild-type (VG982-6A) and pds5-3 (VG988-1C) cells were grown asynchronously at 23°C or shifted to 37°C for 3 h, and then subjected to FACS® analysis to measure DNA content (Materials and Methods). (B) Micrographs showing the mitotic spindle in cells 1 h after release from S phase at 37°C. Strains in A were synchronously released at 37°C from S phase arrest, and, after 1 h, cells were processed for immunofluorescence. Chromosomal DNA (gray) and Tubulin (white). (C) Quantitation of cell and DNA morphologies after release from S phase at 37°C. Strains in B were scored for cell and DNA morphologies in S phase arrested cells at 37°C [S phase (HU)] and in cells 2 h after release at 37°C (2 h after S phase). Data from 400 cells from two independent experiments were scored to generate error bars.
Figure 4
Figure 4
Maintenance of sister chromatid cohesion in wild-type and pds5 cells arrested in mid-M phase. Wild-type (VG982-6A) and pds5-3 (VG988-1C) cells were arrested in G1 phase at 23°C, and then released from G1 phase and arrested in mid-M phase at 23°C. Cells were transferred to 37°C for 1 h while still arrested. Aliquots of mid-M phase cells at 23° and 37°C were plated to determine cell viability (A) and processed for FISH (B). (A) Cell viability. Mid-M phase cells at 23° and 37°C were plated onto YEPD and grown for 3 d at 23°C, and the percentage cell viability was calculated (Materials and Methods). Data was generated from two independent experiments. (B) Sister chromatid cohesion. Mid-M phase cells at 23° and 37°C were processed for FISH using a chromosome XVI centromere distal probe (cosmid 70912). The number of FISH signals in each DNA mass was quantitated as described in Fig. 3 B.
Figure 3
Figure 3
Sister chromatid cohesion wild-type and pds5 cells. (A) Micrographs of mid-M phase cells subjected to FISH using a chromosome XVI CEN-distal probe distal (295 kb from CEN16). Wild-type (VG982-6A) and pds5-2 (VG987-5D) cells were arrested in G1 phase [G1 (αF) 23°C], shifted to 37°C, and then released from G1 and arrested in mid-M phase {M (nocodazole) 37°C}. Mid-M cells were processed for FISH. Chromosomal DNA (gray) and hybridized probe (white). Bar, 5 μm. (B) Quantitation of FISH of G1 and mid-M phase cells. Wild-type (VG982-6A), pds5-1 (VG986-5B), pds5-2 (VG987-5C), and pds5-3 (VG988-1C) were arrested and subjected to FISH as described in A. The number of FISH signals in each DNA mass was determined and plotted as a percentage of total nuclei. 200 nuclei from G1 and mid-M cells were scored to generate data. (C) DNA content of mid-M phase cells. Aliquots of cells from strains in B were fixed and processed for flow cytometry to analyze for DNA content in mid-M phase–arrested cells at 37°C.
Figure 5
Figure 5
Analysis of chromosome condensation in wild-type and pds5 cells. Wild-type (VG982-6A) and pds5-3 (VG988-1C) cells were synchronously arrested in mid-M at 37°C, as described in Fig. 3 A. (A) Micrographs showing FISH of the rDNA. Mid-M phase cells at 37°C subjected to FISH using rDNA as probe. Chromosomal DNA (PI) and hybridized probe (FITC) are shown. Arrows indicate line-like rDNA in wild-type cells. Bar, 5 μm. (B) Micrographs showing FISH of chromosome VIII region. Mid-M phase cells at 37°C subjected to FISH using a mixture of six chromosome VIII probes. Chromosomal DNA (gray) and hybridized probe (white) are shown. Bar, 5 μm.
Figure 7
Figure 7
Localization of Pds5p and Mcd1p in intact yeast cells. Haploid strains VG2063-5C (PDS5-GFP) and VG2049-3A (MCD1-GFP) were grown to mid-log phase at 23°C, and then arrested in G1, S, or mid-M phase. Haploid strains VG2069-1B (cdc15-1 PDS5-GFP) and VG2061-1D (cdc15-2 MCD1-GFP) were grown to mid-log phase at 23°C, and then arrested in telophase by incubation at 37°C for 3 h. Arrested cells were fixed and the localization of GFP-tagged proteins determined in intact cells by fluorescence microscopy (Materials and Methods). Localization of (A) Pds5p and (B) Mcd1p in whole cells at different cell cycle stages.
Figure 6
Figure 6
Cell cycle–dependent localization of Pds5p and Mcd1p to chromosomes. (A) Localization of Pds5p to chromosomes. Strain 2064-2A (PDS5-6HA) was grown at 23°C, and then arrested in either G1, S, or mid-M phase by treatment with α factor, hydroxyurea, or nocodazole, respectively. Aliquots of cells were fixed and processed for chromosome spreads (Materials and Methods). Chromosomal DNA (DAPI) and Pds5p (Pds5-6HAp) are shown. *Anaphase cell from the asynchronous population. Bar, 5 μm. (B) Localization of Mcd1p to chromosomes. Strain VG2050-1A (MCD1-6HA) was grown and processed for chromosome spreads as described in A. Chromosomal DNA (DAPI) and Mcd1p (Mcd1-6HAp) are shown. *Anaphase cell from the asynchronous population. Bar, 5 μm. (C) Enlargement of mid-M phase cells from cultures in A and B. Bar, 1.25 μm. (D) Binding of Pds5p and Mcd1p to CEN3 and LEU2 loci. Strains in A were grown to mid-log phase and subjected to ChIP using the anti–HA antibody C12A5, and then analyzed by PCR for the presence of CEN3, LEU2, and ADE3 chromosomal loci (Materials and Methods). (Total) PCR of total chromatin equivalent to 1% that used for ChIP; positive control for PCR. (E) Assay to determine whether Mcd1p function is required for Pds5p localization to chromosomes. Wild-type (VG2064-2A) and mcd1-1 mutant (VG2072-2D) strains containing Pds5p-6HA were arrested in G1 phase at 23°C, transferred to 37°C, and then arrested in mid-M phase at 37°C and processed for chromosome spreads (Materials and Methods). Chromosomal DNA (DAPI) and Pds5p (Pds5-6HAp) are shown. Bar, 5 μm. (F) Assay to determine whether Pds5p function is required for Mcd1p localization to chromosomes. Wild-type (VG2050-1A) and pds5 mutant ({VG2073-7D; pds5-1}, {VG2074-6C; pds5-2}, and {VG2075-6D; pds5-3} strains bearing Mcd1p-6HA were treated as described in E. Chromosomal DNA (DAPI) and Mcd1p (Mcd1-6HAp) are shown. Bar, 5 μm.
Figure 6
Figure 6
Cell cycle–dependent localization of Pds5p and Mcd1p to chromosomes. (A) Localization of Pds5p to chromosomes. Strain 2064-2A (PDS5-6HA) was grown at 23°C, and then arrested in either G1, S, or mid-M phase by treatment with α factor, hydroxyurea, or nocodazole, respectively. Aliquots of cells were fixed and processed for chromosome spreads (Materials and Methods). Chromosomal DNA (DAPI) and Pds5p (Pds5-6HAp) are shown. *Anaphase cell from the asynchronous population. Bar, 5 μm. (B) Localization of Mcd1p to chromosomes. Strain VG2050-1A (MCD1-6HA) was grown and processed for chromosome spreads as described in A. Chromosomal DNA (DAPI) and Mcd1p (Mcd1-6HAp) are shown. *Anaphase cell from the asynchronous population. Bar, 5 μm. (C) Enlargement of mid-M phase cells from cultures in A and B. Bar, 1.25 μm. (D) Binding of Pds5p and Mcd1p to CEN3 and LEU2 loci. Strains in A were grown to mid-log phase and subjected to ChIP using the anti–HA antibody C12A5, and then analyzed by PCR for the presence of CEN3, LEU2, and ADE3 chromosomal loci (Materials and Methods). (Total) PCR of total chromatin equivalent to 1% that used for ChIP; positive control for PCR. (E) Assay to determine whether Mcd1p function is required for Pds5p localization to chromosomes. Wild-type (VG2064-2A) and mcd1-1 mutant (VG2072-2D) strains containing Pds5p-6HA were arrested in G1 phase at 23°C, transferred to 37°C, and then arrested in mid-M phase at 37°C and processed for chromosome spreads (Materials and Methods). Chromosomal DNA (DAPI) and Pds5p (Pds5-6HAp) are shown. Bar, 5 μm. (F) Assay to determine whether Pds5p function is required for Mcd1p localization to chromosomes. Wild-type (VG2050-1A) and pds5 mutant ({VG2073-7D; pds5-1}, {VG2074-6C; pds5-2}, and {VG2075-6D; pds5-3} strains bearing Mcd1p-6HA were treated as described in E. Chromosomal DNA (DAPI) and Mcd1p (Mcd1-6HAp) are shown. Bar, 5 μm.
Figure 6
Figure 6
Cell cycle–dependent localization of Pds5p and Mcd1p to chromosomes. (A) Localization of Pds5p to chromosomes. Strain 2064-2A (PDS5-6HA) was grown at 23°C, and then arrested in either G1, S, or mid-M phase by treatment with α factor, hydroxyurea, or nocodazole, respectively. Aliquots of cells were fixed and processed for chromosome spreads (Materials and Methods). Chromosomal DNA (DAPI) and Pds5p (Pds5-6HAp) are shown. *Anaphase cell from the asynchronous population. Bar, 5 μm. (B) Localization of Mcd1p to chromosomes. Strain VG2050-1A (MCD1-6HA) was grown and processed for chromosome spreads as described in A. Chromosomal DNA (DAPI) and Mcd1p (Mcd1-6HAp) are shown. *Anaphase cell from the asynchronous population. Bar, 5 μm. (C) Enlargement of mid-M phase cells from cultures in A and B. Bar, 1.25 μm. (D) Binding of Pds5p and Mcd1p to CEN3 and LEU2 loci. Strains in A were grown to mid-log phase and subjected to ChIP using the anti–HA antibody C12A5, and then analyzed by PCR for the presence of CEN3, LEU2, and ADE3 chromosomal loci (Materials and Methods). (Total) PCR of total chromatin equivalent to 1% that used for ChIP; positive control for PCR. (E) Assay to determine whether Mcd1p function is required for Pds5p localization to chromosomes. Wild-type (VG2064-2A) and mcd1-1 mutant (VG2072-2D) strains containing Pds5p-6HA were arrested in G1 phase at 23°C, transferred to 37°C, and then arrested in mid-M phase at 37°C and processed for chromosome spreads (Materials and Methods). Chromosomal DNA (DAPI) and Pds5p (Pds5-6HAp) are shown. Bar, 5 μm. (F) Assay to determine whether Pds5p function is required for Mcd1p localization to chromosomes. Wild-type (VG2050-1A) and pds5 mutant ({VG2073-7D; pds5-1}, {VG2074-6C; pds5-2}, and {VG2075-6D; pds5-3} strains bearing Mcd1p-6HA were treated as described in E. Chromosomal DNA (DAPI) and Mcd1p (Mcd1-6HAp) are shown. Bar, 5 μm.
Figure 6
Figure 6
Cell cycle–dependent localization of Pds5p and Mcd1p to chromosomes. (A) Localization of Pds5p to chromosomes. Strain 2064-2A (PDS5-6HA) was grown at 23°C, and then arrested in either G1, S, or mid-M phase by treatment with α factor, hydroxyurea, or nocodazole, respectively. Aliquots of cells were fixed and processed for chromosome spreads (Materials and Methods). Chromosomal DNA (DAPI) and Pds5p (Pds5-6HAp) are shown. *Anaphase cell from the asynchronous population. Bar, 5 μm. (B) Localization of Mcd1p to chromosomes. Strain VG2050-1A (MCD1-6HA) was grown and processed for chromosome spreads as described in A. Chromosomal DNA (DAPI) and Mcd1p (Mcd1-6HAp) are shown. *Anaphase cell from the asynchronous population. Bar, 5 μm. (C) Enlargement of mid-M phase cells from cultures in A and B. Bar, 1.25 μm. (D) Binding of Pds5p and Mcd1p to CEN3 and LEU2 loci. Strains in A were grown to mid-log phase and subjected to ChIP using the anti–HA antibody C12A5, and then analyzed by PCR for the presence of CEN3, LEU2, and ADE3 chromosomal loci (Materials and Methods). (Total) PCR of total chromatin equivalent to 1% that used for ChIP; positive control for PCR. (E) Assay to determine whether Mcd1p function is required for Pds5p localization to chromosomes. Wild-type (VG2064-2A) and mcd1-1 mutant (VG2072-2D) strains containing Pds5p-6HA were arrested in G1 phase at 23°C, transferred to 37°C, and then arrested in mid-M phase at 37°C and processed for chromosome spreads (Materials and Methods). Chromosomal DNA (DAPI) and Pds5p (Pds5-6HAp) are shown. Bar, 5 μm. (F) Assay to determine whether Pds5p function is required for Mcd1p localization to chromosomes. Wild-type (VG2050-1A) and pds5 mutant ({VG2073-7D; pds5-1}, {VG2074-6C; pds5-2}, and {VG2075-6D; pds5-3} strains bearing Mcd1p-6HA were treated as described in E. Chromosomal DNA (DAPI) and Mcd1p (Mcd1-6HAp) are shown. Bar, 5 μm.
Figure 8
Figure 8
Model for chromosome condensation. (A) Condensation in wild-type cells. (Top) Decondensed chromosome with sister chromatid cohesion. Chromosome condensation begins by either packaging of DNA between cohesion sites (middle left) or by the association of adjacent cohesion sites (middle right). The fully condensed mitotic chromosome (bottom) requires both the association of adjacent cohesion sites and the packaging of the DNA between cohesion sites. (B) Condensation in pds5 or mcd1 mutants. (Top) Decondensed chromosome with dissociated sister chromatids. When chromosomes undergo condensation, the DNA between cohesion sites is still packaged, but the adjacent cohesion sites cannot associate.
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