Figure 3.

Separation of broken DNA ends in the absence of Ku80. (a) Localization of CFP and YFP arrays in the presence and absence of Ku80. Confocal microscopy in control and in Ku80-depleted NIH2/4 cells. Two examples of separated CFP and YFP arrays with different distances are presented. Separation was defined as a tag distance of >500 nm. The scale bars indicate 5 µm in a and c. Arrow indicates ISceI array. (b) Quantification of separated CFP and YFP signals in NIH2/4 cells depleted of the indicated repair factors or in controls cells. Values represent averages ± s.d. (n = 200) from three independent experiments. (c) Partial metaphase spreads of control or Ku80-depleted NIH2/4 cells 24 h after overexpression of HA–ISceI. Localization of the array at the end of a single chromosome 3 indicates the presence of a chromosome break. Arrowheads indicate the L–ISceI–T array. Probes against the entire array were used and the Lac and Tet portions of the array can not be distinguished. (d) Quantification of L–ISceI–T array signals on broken or translocated chromosomes. One hundred metaphases were analysed per sample. (e) Representative CFP–YFP tag separation trajectory in Ku80-depleted NIH2/4 cells. Below the trajectories, coloured bars indicate the disjointness probability (P_D) for every 10-min window (red, P_D <75%; yellow, 75 ≤ P_D ≤ 95%; green, P_D >95%). (f) Distribution of relative fluorescent tag mobility. Boxes indicate boundaries of the 25th percentile to the 75th percentile. The red line indicates the median of the data. Error bars indicate the spread of the data. Outliers are marked by red crosses, and are defined as data points that are further away than 1.5 times the width of the box. P values were obtained by t-test and represent pairwise comparisons of the indicated sample means.