Figure 2.

DarT induces the DDR in TARG1-deficient cells and limits DNA replication (A) Representative images of clonogenic survival in U-2 OS WT or TARG1 KO cells expressing pLIX_403 GFP-DarT WT or GFP-DarT E160A. pLIX_403 expression was controlled by doxycycline. (B) U-2 OS WT or TARG1 KO cells treated with HU (2 mM, 24 h), CPT (1 μM, 1 h), MMS (2 mM, 1 h), DarT WT (24 h), DarT E160A (24 h). Levels of DDR proteins KAP1 pS824, RPA2 pS4/8, γH2AX were analysed in response to the aforementioned genotoxins. (C) QIBC analysis of EdU incorporation in U-2 OS WT and TARG1 KO cells in response to the genotoxins found in Figure 2B. Prior to fixation, cells were treated with EdU (10 μM, 30 min). Red bar represents the mean EdU intensity for each population and each point represents the mean EdU intensity for an individual nuclei. (D) U-2 OS WT or TARG1 KO cells expressing either DarT WT or TARG1 KO for 24 h were pulse labeled with CldU followed by IdU for 25 min each, as indicated by the labeling protocol, top. IdU track lengths were measured for 100 fibers per condition. Statistical tests were performed using ANOVA where ns is not significant and **** is P < 0.0001. In the box plots, the box extends from the 25th (lower edge) to 75th (higher edge) percentiles and the line within the box represents the median. Whiskers extend to the minimum and maximum values recorded. Representative fiber images can be found in Supplementary Figure S2D. (E) U-2 OS TARG1 KO cells expressed GFP-DarT for 24 h and were treated with EdU (10 μM, 30 mins) prior to pre-extraction and fixation. Following a Click-iT reaction to visualise EdU, cells were then immunostained for GFP and chromatin-bound RPA2. Scale bar 20 μm. (F) QIBC analysis of chromatin-bound RPA2 of U-2 OS TARG1 KO cells in response to the genotoxins found in Figure 2B. Blue bar represents the mean RPA2 intensity for each population and each point represents the mean chromatin-bound RPA2 intensity for an individual nuclei.