Raw flow cytometry data

<p>Day: Day of the measure<br> Treatment: Experimental treatment<br> Other columns are the parameters for each object (bacteria)</p

Bacterial doubling time, expressed in hours, as a function of the treatment

<p>Day: Day of the measure (maximal doubling time over 24 hours growth period)<br> Phages: Experimental treatment (see the paper)<br> dt: Doubling time, in hours</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

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

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

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

Pol2 degradation occurs in S phase and is SCF^Pof3 dependent.

<p>Exponentially growing cells (AS) were synchronized at the G₁/S (time zero) boundary in the presence of 12 mM HU for 3 h, and were released into fresh YES medium with or without CHX. Cells were collected and processed for DNA content analysis in <i>A</i>, and for Western blotting in <i>B</i>. (A) Cells were fixed at the indicated times, and DNA contents were analyzed by flow cytometry. (B) Pol2 is degraded during S phase in <i>swi1</i> mutants. Cellular amounts of Pol2-FLAG were determined at the indicated times. Tubulin levels were also monitored as a loading control. Representative results of repeat experiments are shown. (C) Stability of Pol2-FLAG during the CHX treatment shown in <i>B</i> was quantified as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003213#pgen-1003213-g001" target="_blank">Figure 1</a>. For each strain, relative intensity of the Pol2-FLAG band at 0 h was set to 1. (D) Pol2 is stabilized in the <i>skp1-94</i> mutants. Exponentially growing <i>skp1-94</i> cells were treated with CHX at 25°C and 35°C. Cellular amounts of Pol2-FLAG were examined from 0 to 4 h of CHX treatment. (E) <i>pof3</i> deletion stabilizes Pol2. As in <i>D</i>, Pol2-FLAG levels were examined during the CHX treatment of wild-type and <i>pof3</i>Δ cells. (F) Pof3 interacts with Pol2. Cells expressing the indicated fusion proteins (Pol2-FLAG and/or Pof3-Myc) were harvested in the presence or absence of DSP, and protein extracts were prepared. Pof3-Myc was immunoprecipitated, and associated proteins were probed with anti-FLAG antibody. Representative results of repeat experiments are shown. IP, immunoprecipitation; WB, Western blotting; WCE, whole cell extract.</p