Nbs1-S343A mutant disrupts the intra–S-phase checkpoint, inhibits HRR, and down-regulates XIAP. B/A-M07e cells expressing GFP (empty) or GFP and Nbs1-S343A mutant (Nbs1-S343A) were characterized in Figure 4A (B/A-GFP empty and B/A-GFP Nbs1-S343A cells, respectively). (A) RDS assay. Results show the ratio of ³H-thymidine to ¹⁴C-thymidine normalized to the corresponding untreated control to determine the relative amount of DNA synthesis. Results are representative of 5 experiments. *P < .05. (B) Cells were untreated or treated with 0.25 μg/mL MMC and cell-cycle distribution was analyzed 0, 12, and 24 hours later by flow cytometry after staining with propidium iodide. Results represent 3 independent experiments. *P < .05 in comparison to corresponding S-phase in empty group. (C) NHEJ-mediated end-ligation of the XhoI + XbaI–digested plasmid substrate (monomers) by the lysis buffer (Control) or cell lysates from B/A-GFP empty and B/A-GFP Nbs1-S343A cells generated multiplasmid products (dimers, trimers). The mean percentages of repair products (dimmer, trimer) (± SD) are shown below the lanes. (D) HRR-mediated repair of I-SceI–induced DSBs in DR-GFP cassette in B/A-GFP empty and B/A-GFP Nbs1-S343A cells could be evaluated by semiquantitative PCR generating products from repaired and unrepaired (substrate) DR-GFP cassette. Control consisted of cells carrying only unrepaired DR-GFP cassette. The mean percentages of repair product (± SD) are shown below the lanes. *P < .05. (E) XIAP and Bcl-xL expression was examined by Western blot analysis in total cell lysates; tubulin served as loading control.