Figure 3.

E160D MEF cells had more collapsed replication forks and lower DNA replication rates in response to DNA damage. (A) and (B) Replication tracks of WT, WT/E160D and E160D/E160D cells in the absence or presence of H₂O₂. (A) Outline for labeling newly synthesized DNA with IdU (green) and CldU (red) and treatment with H₂O₂. Images indicate the typical undamaged replication fork (green/red track) and collapsed replication fork (green-only track). Red-only track that would represent the newly fired replication fork is not shown. (B) Representative images of DNA fibers of WT and E160D cells untreated or treated with H₂O₂. In left bottom panels, yellow arrows specified the normal replication forks (green/red tracks) without DNA damage, and white arrows indicated the stalled replication forks due to DNA damage (green only tracks). Replication tracks were assessed and quantified and the relative level of collapsed replication forks in each sample was expressed as the percentage of the total number of incorporated DNA forks (all tracks). Values are means ± s.d. of three independent experiments (Right panel). (C) DNA replication rates of WT, WT/E160D and E160D/E160D cells under normal conditions. MEF cells were cultured in DMEM medium with 1 μCi/ml [³H] thymidine, and were collected after a specific time (0, 2, 5, or 8 h). Cells were extensively washed and lysed. DNA was precipitated and the radioactivities of all DNA samples were counted by a liquid scintillation counter. Values are means ± s.d of three independent assays. (D) MEF cells were treated (or not) with MNU (200 μg/ml, 1h) or H₂O₂ (1 mM, 15 min). Cells were then incubated in DMEM medium containing 1 μCi/ml [³H] thymidine (5 h). The incorporation of [³H] thymidine was quantified and the relative [³H] thymidine incorporation rate of each DNA sample was calculated. The incorporation rate of corresponding untreated cells was arbitrarily set as 100.