DDT domain is essential for Swi1’s functions.

<p>(A) Five-fold serial dilutions of the indicated cells were incubated on YES agar medium supplemented with the indicated drugs for 2 to 4 days at 32°C. Representative images of repeat experiments are shown. (B) Cells of indicated <i>swi1</i> mutants were engineered to express Rad22-YFP and grown in YES medium at 25°C until midlog phase. The percentages of nuclei with at least one Rad22-YFP focus are shown (left panel). At least 200 cells were counted for each strain. Error bars correspond to standard deviations obtained from at least three independent experiments. Quantification of Rad22-YFP foci according to the cell cycle stages was also performed by analyzing cell length, nuclei number and position, and the presence of a division plate, as described in our previous publications (right panel) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043988#pone.0043988-Noguchi1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043988#pone.0043988-Noguchi2" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043988#pone.0043988-Ansbach1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043988#pone.0043988-Noguchi4" target="_blank">[43]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043988#pone.0043988-Noguchi6" target="_blank">[55]</a>. Schematic drawing for nuclear and morphological changes during the <i>S. pombe</i> cell cycle is shown (right panel). <i>swi1-h1</i> and <i>swi1</i>^Δ<i>DDT</i> cells displayed strong accumulation of Rad22-YFP foci during S and early G2 phases.</p

Plasmids used in this study.

*<p>The <i>S. pombe nmt1</i> promoter for overpexpression of GST- or FLAG-fused proteins.</p

Swi1 facilitates the recruitment of Swi3 to chromatin.

<p>(A, B) ChIP assays of the indicated Swi1 truncated mutants were performed. Swi1 full-length (1–971), Swi1 (250–550), and Swi1 (250–550) precipitate the <i>ori2004</i> regions (<i>ori2004</i> and two positions 14- and 30-kb away from <i>ori2004</i>) in wild-type (WT, in A, middle panels) and <i>swi3</i>Δ mutants (in B, middle panels). Western blotting with anti-FLAG antibodies shows that all FLAG-fused Swi1 truncations mutants were similarly expressed (top panels). Fold increase in chromatin association over background (FLAG, set to 1) was calculated for each band. The average fold increase in the association of Swi1 truncations with the three positions (<i>ori200</i>4, −14 kb, and −30 kb) is shown, and error bars represent standard deviations obtained from the three positions (bottom panels). Representative results of repeat experiments are shown. WCE, whole cell extract; WB, Western blotting. (C) ChIP assay of GST-Swi3 was performed in wild-type or <i>swi1</i>Δ cells expressing the indicated proteins. GST-Swi3 strongly associates with the <i>ori2004</i> region in wild-type (WT), while GST-Swi3 has weak chromatin association in the absence of Swi1. Western blotting with anti-GST antibodies shows that GST-Swi3 was expressed similarly in wild-type and <i>swi1</i>Δ cells. Quantification of PCR bands was performed as described above. Fold increase in chromatin association over background (GST, set to 1) in each cell line (WT or <i>swi1</i>Δ) is shown. Representative results of repeat experiments are shown. (D) In vitro DNA binding assays of Swi1 (235–564) and Swi3 purified from the indicated <i>S. pombe</i> cells (top panel). The indicated proteins were mixed with radiolabeled plasmid pUC28, and associated DNA was analyzed by agarose gel electrophoresis (middle panel). Quantification of bound DNA was performed as described in Materials and Method. The values of bound DNA were normalized to the amount of proteins used in the reactions, and relative levels of bound DNA over background (GST) are shown. Representative results of repeat experiments are shown. C, input radiolabeled DNA.</p

The structure of the <i>S. pombe</i> Swi1 protein.

<p>(A) The Swi1 polypeptide was divided into 9 putative functional sub-domains. Hatched boxes indicate the regions with amino acid sequences that are conserved throughout evolution. Swi1 contains the Timeless domain (22–279 aa), NLS (304–314 aa), the DDT domain (323–378 aa) and the Timeless-C domain (595–817 aa). h1, h2 and h3 in the DDT domain indicate alpha-helix regions. Swi1 also has stretches of acidic amino acids at 535–542, 858–867, and 916–924 aa regions. The four truncated versions of Swi1 constructed in this study are shown. aa, amino acid. (B) Multiple sequence alignment of DDT domains of various transcription factors, chromatin-remodeling proteins, human Tim1 (Timeless) and <i>S. pombe</i> Swi1. Conserved aromatic and hydrophobic residues are shown in red. The predicted helices are boxed. Asterisks indicate mutated amino acids in <i>swi1</i> mutants constructed in this study.</p

Domains of Swi1 required for Swi1-Swi3 complex formation.

<p>The indicated Swi1 truncations mutants fused to FLAG were expressed in and purified from <i>swi3</i>Δ cells (top panel). Anti-FLAG agarose beads bound to the indicated Swi1 truncation mutant were incubated with recombinant His₆-Swi3. The beads were washed and analyzed by Western blotting using the anti-FLAG or His₆ antibody (middle panel). Asterisks indicate non-specific bands. Quantification of His₆-Swi3 bands was performed using EZQuant, normalizing the values to the amounts of Swi1 truncations used in the reactions. Swi3 binding activity of Swi1 (1–971) was set to 1. Representative image of repeat experiments are shown.</p

<i>S. pombe</i> strains used in this study.

*<p>All strains are <i>leu1-32</i> and <i>ura4-D18</i>.</p

Coordinated Degradation of Replisome Components Ensures Genome Stability upon Replication Stress in the Absence of the Replication Fork Protection Complex

<div><p>The stabilization of the replisome complex is essential in order to achieve highly processive DNA replication and preserve genomic integrity. Conversely, it would also be advantageous for the cell to abrogate replisome functions to prevent inappropriate replication when fork progression is adversely perturbed. However, such mechanisms remain elusive. Here we report that replicative DNA polymerases and helicases, the major components of the replisome, are degraded in concert in the absence of Swi1, a subunit of the replication fork protection complex. In sharp contrast, ORC and PCNA, which are also required for DNA replication, were stably maintained. We demonstrate that this degradation of DNA polymerases and helicases is dependent on the ubiquitin-proteasome system, in which the SCF^Pof3 ubiquitin ligase is involved. Consistently, we show that Pof3 interacts with DNA polymerase ε. Remarkably, forced accumulation of replisome components leads to abnormal DNA replication and mitotic catastrophes in the absence of Swi1. Swi1 is known to prevent fork collapse at natural replication block sites throughout the genome. Therefore, our results suggest that the cell elicits a program to degrade replisomes upon replication stress in the absence of Swi1. We also suggest that this program prevents inappropriate duplication of the genome, which in turn contributes to the preservation of genomic integrity.</p> </div

Ubiquitin-proteasome-dependent degradation of replisome core components in the absence of Swi1.

<p>(A) Inactivation of the proteasome stabilizes Pol2. Cells of the indicated genotypes were incubated for 2 h at the indicated temperatures, then treated with CHX for 4 h. Cellular levels of Pol2-FLAG were monitored after 0, 2 and 4 h of CHX treatment. Tubulin was used as a loading control. (B) Cellular levels of Pol3-FLAG and Mcm6-GFP in the indicated cells were determined after 0 and 4 h of CHX treatment as described in A. (C) Stability of Pol2-FLAG, Pol3-FLAG and Mcm6-GFP at 25°C shown in A and B was quantified. Relative intensity of protein bands at 0 h in each cell type was set to 1. Error bars correspond average deviation (<i>n</i> = 2) or standard deviation (<i>n</i> = 3). (D) Pol2 and Pol3 are highly ubiquitinated in the absence of Swi1. 6xHis-Ub peptide was expressed 22 hours in the absence of thiamine (−B1) at 25°C, then cells were placed for 2 hours at 25°C or 35°C. There is some leaking expression of 6xHis–Ub before induction in the presence of thiamin (+B1). Ubiquitinated proteins were purified as described in Materials and Methods. Western blotting of the indicated protein was performed. W, whole cell extract. Representative results of repeat experiments are shown.</p

Swi1 prevents degradation of DNA polymerases and helicases.

<p>Exponentially growing cells were treated with 0.1 mg/ml CHX at 25°C. (A) Cellular amounts of Pol2-FLAG and Pol3-FLAG were examined from 0 to 3 h of CHX treatment. The anti-FLAG M2 antibody was used to detect Pol2 and Pol3. Western blotting of tubulin was also performed as a loading control. (B) Stability of Pol2-FLAG and Pol3-FLAG shown in <i>A</i> was quantified by NIH ImageJ. Relative intensity of protein bands at 0 h was set to 1 in each experiment. Error bars correspond to standard deviation of three independent experiments. <i>wt</i>, in blue; <i>swi1</i>Δ, in red. (C) Cellular amounts of Mcm2-GFP, Mcm6-GFP, Mcm4, Mrc1, Orc1-FLAG, and PCNA were determined from 0 to 4 h of CHX treatment. Anti-FLAG, anti-GFP, anti-Mcm4, anti-Mrc1, and anti-PCNA antibodies were used for Western blotting. (D) Stability of Mcm2-GFP, Mcm6-GFP, Mcm4, and Mrc1, shown in <i>C</i>, was quantified as described in <i>B</i>. Error bars represent average deviation (<i>n</i> = 2) or standard deviation (<i>n</i> = 3). (E) Replisome components in a chromatin-enriched fraction were degraded in response to CHX. Chromatin-free (Triton-soluble) and chromatin-enriched (Triton-insoluble) fractions were prepared from <i>S. pombe</i> cells treated with CHX for 0 and 4 h. The fractions were analyzed by Western blotting using antibodies to detect the indicated proteins.</p

Forced accumulation of replisome components in <i>swi1</i>Δ cells causes catastrophic DNA replication and mitotic abnormalities.

<p>(A) <i>swi1</i>Δ <i>pof3</i>Δ cells have increased levels of mitotic catastrophes. Exponentially growing cells were treated with or without the indicated drugs (12 mM HU or 20 µM CPT for 6 h), fixed in ethanol, and stained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images of observed nuclear phenotypes are shown. Representative mitotic failures are shown by arrows. Arrows were omitted from the images of <i>swi1</i>Δ <i>pof3</i>Δ cells because a large numbers of cells showed mitotic catastrophes. The scale bar represents 10 µM. (B) Quantification of cells with defective mitosis including chromosome missegregation, aneuploidy, cut and other aberrant phenotypes. More than 200 cells were counted for each strain. Error bars correspond to standard deviations obtained from three experiments. (C) DNA damage sensitivity of <i>swi1</i>Δ mutants is increased by <i>pof3</i> deletion. Five-fold serial dilutions of cells were incubated on YES agar medium supplemented with the indicated drugs (2 mM HU or 1 µM CPT) for 2 to 3 days at 32°C. (D) <i>pof3</i> deletion exacerbates replication recovery defects of <i>swi1</i>Δ mutants. Exponentially growing cells (Log) were incubated in the presence of 5 µM CPT for 3 h at 30°C (CPT), then washed and returned into fresh medium for 2 h or 4 h (2 h, 4 h). Chromosome samples were examined by PFGE. Representative results of repeat experiments are shown. <i>swi1</i>Δ cells have shorter chromosome III due to hyper recombination at rDNA repeats <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003213#pgen.1003213-Sommariva1" target="_blank">[42]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003213#pgen.1003213-Rapp1" target="_blank">[70]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003213#pgen.1003213-Ansbach1" target="_blank">[101]</a>.</p