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.