Figure 4(A) Effect of the rad54∆ and rad52∆ mutations in spontaneous and MMS-induced uSCE. The frequency of spontaneous and MMS-induced recombinants was determined in mid-log phase cell cultures before (spontaneous uSCE) and after treatment with 0.02% MMS for 4 hours (DNA damage-induced uSCE). Thus, the frequency of DNA damage-induced uSCE includes the recombinants that arose spontaneously before MMS addition. The frequency of uSCE was determined at 0.02% MMS because at higher MMS concentrations the loss of viability of rad52∆ and rad54∆ mutants did not allow us to select for His+ recombinants. The mean and SEM of 5 independent fluctuation tests are shown; n.a., not applicable. Data information: Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. (B) ssDNA repair efficiency of the indicated mutants as determined from the maximal and final percentages of cells with RPA foci during the time course (See Figures EV2D-E for the time course analyses; cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times). Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. Number of foci per cell in cells with foci (C) at times 1 or 2 (peak) and 7 hours from the time course shown in (B). Counting and quantification were done with the MetaMorph software. Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. fluorescence signal per focus (D) at times 1 or 2 (peak) and 7 hours from the time course shown in (B). Representative images of wild-type and mutant cells with MMS-induced RPA foci (bright field and fluorescence signal) are shown (D; left panels). Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. fluorescence signal of RPA foci per cell (E) at times 1 or 2 (peak) and 7 hours from the time course shown in (B). Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. (F) ssDNA repair efficiency of the indicated mutants as determined by ChIP against Rfa1-YFP at the ARS305 replication origin. Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium. The amount of Rfa1 at ssDNA was determined at 1 and 7 hours after MMS release. RPA enrichment was calculated as the ratio between immunoprecipitate and input values. All values were normalized to the wild type at 7 hours, taken as 1. Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. In (F) all values were significant relative to the untagged controls (not shown for clarity).