DNA polα mutant, <i>swi7H4</i> exhibit conditional lethality with <i>chk1</i> knockout.

<p>(A) Indicated strains were grown at 25°C, serially diluted and spotted on YEA plates. Plates were incubated at indicated temperature for 3 days before taking photographs. (B) Percent Survival of <i>swi7H4 chk1</i> knockout strain at non permissive temperature: Indicated strains were grown till mid log phase at 25°C then shifted at 36°C. Sample were collected at 1.5 hour intervals, equal number of cells were plated on YEA plates and incubated at 25°C. Number of surviving colonies was calculated as the percentage of colonies, appearing at permissive temperature. Values shown are the average of three independent experiments with standard deviation. (C) The plates showing surviving colonies from figure B at time point 0 (upper panel) and 6hr at 36°C (lower panel) were photographed.</p

Genetic interaction studies of <i>swi7H4</i> mutant with genes involve in DNA repair pathway.

<p>Indicated strains were grown at 25°C, serially diluted and spotted on YEA plate. Plates were incubated at indicated temperature for 3–4 days before taking photographs.</p

Strains used in this study.

<p>Strains used in this study.</p

<i>swi7H4</i> mutant cells delay the mitotic entry and activates checkpoint kinase Chk1.

<p>(A) Cells were processed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124063#pone.0124063.g003" target="_blank">Fig 3</a>, samples were collected at 15 minute interval and the number of cells having two nuclei was counted using DAPI as an indication of cells entering into mitosis. (B) Indicated strains were grown at permissive temperature till mid log phase then shifted at 36°C for indicated time. Protein lysate was prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124063#sec002" target="_blank">Material and Methods</a>, samples were run on 8% SDS PAGE, transferred on nitrocellulose membrane and probed with anti HA antibody.</p

<i>swi7H4</i> mutant cells exhibit chromosome segregation defect at non permissive temperature.

<p>(A) Cells were grown at 25°C till mid log phase and then shifted at 36°C for 6 hr, fixed with 70% ethanol and stained with DAPI to visualize nuclei (upper panel) or with differential interference contrast (DIC) filter (lower panel). Arrow indicates defective chromosome segregation. (B) Indicated strains were grown till mid log phase at 25°C and then shifted at 36°C. Cells were collected at indicated time interval, fixed with 70% ethanol and stained with DAPI. Cells having aberrant chromosome segregation were counted.</p

Mitotic progression was delayed in <i>swi7H4</i> mutant cells.

<p>Strains were grown till mid log phase, synchronized in early S phase by treating with 12mM hydroxyurea (HU) for 4 hr at 25°C, washed and released at 36°C. Samples were collected at 30 minute time interval fixed, stained with propidium iodide and FACS analysis was performed using BD FACS calibre for DNA content analysis.</p

<i>swi7H4</i> mutant cells have elevated level of Rad22 YFP foci at non permissive temperature.

<p>(A) Indicated strains were grown till mid log phase at 25°C, shifted at non permissive temperature (36°C) for 6hr. The cells were processed for indirect immunofluorescence microscopy as described in <i>Materials and Methods</i>. (B) About 200 cells for each sample were counted and the percentage of cells containing Rad22-YFP foci was plotted.</p

The diploidisation of <i>wat1-17</i> and <i>wat1-17 chk1Δ</i> strain.

<p><b>A.</b> Wild type, <i>wat1-17</i>, <i>chk1Δ</i> and <i>wat1-17chk1</i>Δ double mutant were grown up to mid log phase, about 1000 cells were spread on YEA plates containing 1.5 µg/ml Phloxine B. All the plates were incubated at 25°C for 3–4 days before taking photographs. <b>B.</b> FACS analysis of wild type, <i>chk1</i>Δ, <i>wat1-17, wat1-17chk1</i>Δ mutants. The asynchronous cultures were grown at 25°C and shifted to 18°C, samples were taken at 12 h interval, fixed and stained with the propidium iodide. Samples were analyzed for BD FACS caliber for DNA content analysis.</p

The <i>ts17/wat1-17</i> mutant allele exhibit conditional lethality with <i>chk1</i> knockout.

<p>Indicated strains were grown at 25°C, serially diluted and spotted on YEA plates. Plates were incubated at indicated temperature for 3 days except 18°C plate that was incubated for 7 days before taking photographs.</p

Mapping of <i>wat1-17</i> mutation and its conservation with human Lst8.

<p><b>A.</b> Location of mutation in <i>wat1-17</i> gene. <b>B.</b> ESPript generated sequence alignment of Wat1 and human Lst8. Secondary structure assignment was according to crystal structure Lst8 (PDB-ID, 4JSP).</p