The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci - PubMed (original) (raw)

. 2000 Jan 1;14(1):81-96.

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The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci

C Frei et al. Genes Dev. 2000.

Abstract

We have examined the cellular function of Sgs1p, a nonessential yeast DNA helicase, homologs of which are implicated in two highly debilitating hereditary human diseases (Werner's and Bloom's syndromes). We show that Sgs1p is an integral component of the S-phase checkpoint response in yeast, which arrests cells due to DNA damage or blocked fork progression during DNA replication. DNA polepsilon and Sgs1p are found in the same epistasis group and act upstream of Rad53p to signal cell cycle arrest when DNA replication is perturbed. Sgs1p is tightly regulated through the cell cycle, accumulates in S phase and colocalizes with Rad53p in S-phase-specific foci, even in the absence of fork arrest. The association of Rad53p with a chromatin subfraction is Sgs1p dependent, suggesting an important role for the helicase in the signal-transducing pathway that monitors replication fork progression.

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Figures

Figure 1

Figure 1

sgs1 cells are hypersensitive to HU but not to UV and γ radiation. (A–C) A total of 200 m

m

HU was added to exponentially growing cells and aliquots were washed and plated in triplicate on rich medium without HU at the indicated times. After 2–3 days, viability was scored. Standard deviation was <5%; for simplicity; error bars are not shown. All strains are isogenic to GA-871 carrying the following indicated mutations: (♦) wild type (GA-871); (□) sgs1::LEU2 (GA-880); (▴) pol2-11 (GA-1021); (▵) pol2-11 sgs1::LEU2 (GA-1022); (×) pol2-11 rad24::TRP1 (GA-1023); (●) rad24::TRP1 (GA-872); (█) rad24::TRP1 sgs1::LEU2 (GA-881). A and C were performed at 25°C (permissive temperature for the pol2-11 allele). B was performed as A, but the cells were incubated in HU at 28°C (semirestrictive temperature for pol2-11 allele), and plated at 25°C. (D) HU sensitivity of sgs1 cells depends on the helicase activity. sgs1::hisG (GA-737) were transformed with the empty vector (pRS415; □), p_SGS1_ (pJL31; ♦), p_sgs1_-hd (helicase mutant; pJL37; ○) or p_sgs1_-ct (carboxy-terminal truncation; pJM502; star). Plasmids are described in Lu et al. (1996). HU was added and viability was scored as above. (E) sgs1::LEU2 cells are partially defective for the S/M checkpoint. Cells were arrested in G1 with α-factor and released into S phase with (right) or without 200 m

m

HU (left). Aliquots were taken at the indicated times, stained with DAPI, and scored for cells that have undergone nuclear division. Staining of mitotic spindles with an antitubulin antibody gives an identical percentage of postanaphase cells (data not shown). FACS analysis shows that >98% of the cells fail to complete DNA replication during the course of the experiment (data not shown). Symbols and strains as in A–C. (F) sgs1::hisG cells are not hypersensitive to UV light. Cells were plated, UV radiated with the indicated dose, and plated on rich medium. (♦) SGS1 (GA-59); (□) sgs1::hisG (GA-737). (G) sgs1::hisG cells are not hypersensitive to γ radiation. Cells were plated and radiated with the indicated dose. All strains in D, F, and G are isogenic.

Figure 2

Figure 2

sgs1::LEU2 cells are partially defective for the intra-S checkpoint. (A) Exponentially growing cells were grown at 30°C and blocked in G1 with α-factor for two-thirds of the generation time. α-factor was washed away and the cells were incubated in fresh medium containing 0.033% MMS. Aliquots for FACS analysis were taken after the indicated time. The 1N and 2N DNA content peaks were taken from time point 0 and 30 min (without MMS) and indicated for every FACS sample as small bars at the top of each panel. In no case, were peaks other than 1N and 2N observed. Isogenic strains were as follows: wild type (GA-871); sgs1::LEU2 (GA-880); rad24::TRP1 (GA-872); rad24::TRP1 sgs1::LEU2 (GA-881); mec1 (mec1-1 sml1; GA-904); The end of S phase in the presence of MMS is indicated by an arrow. (B) The same as in A but all manipulations were done at 25°C, which allows growth of pol2-11. The time required to finish S phase as determined by a FACS scan is indicated. Note that the pol2-11 mutants traverse S phase more slowly than wild type in the absence of DNA damage (Budd and Campbell 1993). pol2-11 (GA-1021); pol2-11 sgs1::LEU2 (GA-1022); pol2-11 rad24::TRP1 (GA-1023), and pol2-11 rad24::TRP1 sgs1::LEU2 (GA-1024).

Figure 3

Figure 3

sgs1::LEU2 cells are proficient for the G2/M (A) and G1/S (B) DNA damage checkpoints. (A) Exponentially growing cells in YPAD were arrested at G2/M with 15 μg/ml nocodazole for 2 hr. For the last 30 min of the block, 0.15% MMS was added to one-half of the culture. MMS was then inactivated by the addition of 1 vol 10% Na-thiosulfate, cells were then washed and released in medium lacking nocodazole and MMS. After the indicated times, aliquots were removed, stained with DAPI, and cells of each strain were scored microscopically separated chromatids (post-anaphase). Strains isogenic to GA-871 were as follows: (♦) Wild type (GA-871); (□) sgs1::LEU2 (GA-880); (●) rad24::TRP1 (GA-872); (█) rad24::TRP1 sgs1::LEU2 (GA-881). (B) Exponentially growing cells were blocked in G1 with α-factor for 70 min and 0.2% MMS was added to one-half of the culture and incubated for the last 10 min of the α-factor block. The cells were then washed and released into medium lacking both α-factor and MMS. Aliquots were taken after the indicated time and FACS analysis was performed. Arrows indicate the aliquot when the cells enter S phase. These mutant strains are isogenic with wild type (GA-871) and contain full deletions or disruptions as described in A.

Figure 4

Figure 4

SGS1 and RAD24 are both required for Rad53p phosphorylation in HU. (A) Exponentially growing cells were blocked in G1 with α-factor for two-thirds of the generation time. α-factor was inactivated by the addition of pronase (50 μg/ml; Sigma) and HU was added to 200 m

m

. Aliquots were removed from the exponential growing cultures and at 0, 60, 80, and 100 min after release. Total cell extracts were prepared, run on a 7.5% SDS–polyacrylamide gel and blotted with the 9E10 antibody (α-Myc) for Rad53p–Myc. Wild-type extracts from an exponential culture and from cells 100 min after release were run on every gel and are shown in the last two slots (underlined). Isogenic strains with indicated mutations were as follows: wild type (GA-1040); sgs1::LEU2 (GA-1041); rad24::TRP1 (GA-1042); rad24::TRP1 sgs1::LEU2 (GA-1043); mec1-1 sml1 (GA-1048). The slower migrating band is the phosphorylated form of Rad53p. (B) Western blots were scanned and the percentage of phosphorylated Rad53p was quantified (Aida 200). (C) Aliquots were removed after the indicated time and after release into HU, fixed with Na-azide (0.0325%; Fluka), and budding index was scored under a light microscope. (♦) wild type; (□) sgs1::LEU2; (●) rad24::TRP1; (█) rad24::TRP1 sgs1::LEU2; (+) mec1-1.

Figure 5

Figure 5

Sgs1p protein levels peak in S phase and Sgs1p localizes to nuclear foci. Diploid _cdc16-1 SGS1_-Myc strain (GA-1151) was blocked at the metaphase–anaphase transition by shifting to nonpermissive temperature (36°C) for 2 hr. Cells were released by shifting back to 25°C. Aliquots were removed at the indicated times postrelease for total cell extracts, scoring of the budding index (A), and for immunofluorescence (B). (A) Total cell extracts were run on an 8% SDS–polyacrylamide gel and blotted for Sgs1p–Myc. Sgs1p was quantified and normalized with tubulin. The budding index (♦) indicates re-entry into S phase. (B) Sgs1p–Myc was detected as described in Materials and Methods with 9E10 (green) and cells were counterstained for the abundant nucleolar antigen Nop1p (red; Gotta et al. 1997). A single 0.4 μm confocal microscope section is shown and colocalization is indicated in yellow. A control strain lacking the Myc epitope (GA-174) is shown for HU-arrested cells in the inset of HU blocked cells. For higher resolution analysis, 30–40 Z sections of 0.1-μm were captured on identical stained S-phase cells. Deconvolution was done with Huygans and the staining was reconstituted in 3 dimensions (Bitplane; see Materials and Methods). Colocalization is shown in yellow (bottom). Double labeling of Sgs1–Myc in the diploid strain GA-1151 was performed with anti-Myc (9E10) and an affinity-purified chicken anti-Sgs1p (Sinclair et al. 1997; bottom, last three pictures), with distinct fluorescent markers. Regions in which the two staining patterns coincide are shown in the bottom right panel. Bar, 2 μm.

Figure 5

Figure 5

Sgs1p protein levels peak in S phase and Sgs1p localizes to nuclear foci. Diploid _cdc16-1 SGS1_-Myc strain (GA-1151) was blocked at the metaphase–anaphase transition by shifting to nonpermissive temperature (36°C) for 2 hr. Cells were released by shifting back to 25°C. Aliquots were removed at the indicated times postrelease for total cell extracts, scoring of the budding index (A), and for immunofluorescence (B). (A) Total cell extracts were run on an 8% SDS–polyacrylamide gel and blotted for Sgs1p–Myc. Sgs1p was quantified and normalized with tubulin. The budding index (♦) indicates re-entry into S phase. (B) Sgs1p–Myc was detected as described in Materials and Methods with 9E10 (green) and cells were counterstained for the abundant nucleolar antigen Nop1p (red; Gotta et al. 1997). A single 0.4 μm confocal microscope section is shown and colocalization is indicated in yellow. A control strain lacking the Myc epitope (GA-174) is shown for HU-arrested cells in the inset of HU blocked cells. For higher resolution analysis, 30–40 Z sections of 0.1-μm were captured on identical stained S-phase cells. Deconvolution was done with Huygans and the staining was reconstituted in 3 dimensions (Bitplane; see Materials and Methods). Colocalization is shown in yellow (bottom). Double labeling of Sgs1–Myc in the diploid strain GA-1151 was performed with anti-Myc (9E10) and an affinity-purified chicken anti-Sgs1p (Sinclair et al. 1997; bottom, last three pictures), with distinct fluorescent markers. Regions in which the two staining patterns coincide are shown in the bottom right panel. Bar, 2 μm.

Figure 6

Figure 6

Sgs1p colocalizes with Rad53p and replication foci, but not with Rap1p. (A) A MATa/a diploid Sgs1p–Myc-tagged strain (GA-878) was blocked in G1 with α-factor and released into S phase. Immunofluorescence was performed as described in Materials and Methods by double labeling for Sgs1p–Myc (9E10) and Rad53p (rabbit anti-Rad53p). As a control, a diploid Rad53p–Myc-tagged strain (GA-1126) was double labeled with 9E10 (green) and affinity-purified rabbit anti-Rad53p (red), inset. The signal of Rad53p overlaps precisely with either Myc-tagged protein and is shown in the panel labeled Coloc. Z sections for three-dimensional reconstitution were taken and deconvolved as in Fig. 5B, but only one focal section of the reconstituted image is shown. In the merge of Sgs1p–Myc (9E10; green) and rabbit anti-Rad53p (red); the colocalization is yellow. (B) Temperature-sensitive diploid _cdc4-3/cdc4-3 ORC1_–HA/_ORC1_–HA cells (GA-1222) were blocked in G1 by shifting to 36°C for 2 hr. Cells were released by shifting back to permissive temperature (23°C) for 1 hr until cells entered S phase as judged by FACS analysis (data not shown). Double labeling was performed with HA.11 antibodies (Orc1–HA, green) and rabbit anti-Rad53p (red). (C) S-phase cells (as in A) were double labeled for Sgs1p–Myc (9E10) and goat anti-Orc2p (Santa Cruz, CA). Potential background staining by Orc2p antiserum was eliminated by a preincubation with the antigenic Orc2p peptide (inset). Because of the weak staining of the goat anti-Orc2p antiserum, we could not perform three-dimensional reconstitution. (D) Double labeling was performed with rabbit anti-Rap1p (red) and Sgs1p–Myc (9E10, green) in GA-878 released into S phase as in A. A single focal section of the deconvolved image is shown as in A. Overlapping regions are yellow. Bar, 2 μm.

Figure 7

Figure 7

Rad53p cofractionates with Sgs1p in an insoluble chromatin fraction. (A) Scheme of the cell fractionation assay (left; see Materials and Methods for details). A random culture of GA-990 (Sgs1p–Myc) was used. One volume, representing the same number of starting cells for WCE and Sup samples, and fivefold equivalent volumes of Chr and Sc were used for Western blot analysis with different antibodies. Antibodies used were as follows: 9E10 for Sgs1p–Myc; anti-tubulin (gift of V. Simanis, ISREC) and mAb414 for Nup97 (BAbCO, Berkeley CA), while rabbit sera included, anti-Rad53p (gift of M. Schwartz), anti-topo II (Klein et al. 1992), anti-histone H3 (gift of D. Allis, University of Virginia), and anti-Rfc3p (gift of P. Burgers). All detections shown are performed sequentially on one membrane except histone H3, Rfc3p, and tubulin, which are from a parallel experiment. Bradford readings were taken to determine the protein concentration in each fraction, and are shown as percent of total protein per fraction. (B) _SGS1_–Myc (GA-990) and sgs1::hisG (GA-737) cells were blocked in G1 with α-factor and released into S phase. Cell fractionation was performed and blotted as in A. (Arrow) topo II; (●) an unidentified cross-reacting protein detected by the anti-topo II serum. (C) Western blots from B were quantified and normalized with pore staining and the distribution of each protein in the different fractions is shown as percent of total protein.

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References

    1. Aparicio OM, Weinstein DM, Bell SP. Components and dynamics of DNA replication complexes in S. cerevisiae: Redistribution of MCM proteins and Cdc45p during S phase. Cell. 1997;91:59–69. - PubMed
    1. Araki H, Leem SH, Phongdara A, Sugino A. Dpb11, which interacts with DNA polymerase II(ε) in S. cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint. Proc Natl Acad Sci. 1995;92:11791–11795. - PMC - PubMed
    1. Auerbach AD, Verlander PC. Disorders of DNA replication and repair. Curr Opin Pediatr. 1997;9:600–616. - PubMed
    1. Bierne H, Michel B. When replication forks stop. Mol Microbiol. 1994;13:17–23. - PubMed
    1. Budd ME, Campbell JL. DNA polymerases δ and ε are required for chromosomal replication in S. cerevisiae. Mol Cell Biol. 1993;13:496–505. - PMC - PubMed

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