Figure 3.
PrimPol Is Required for Replication Fork Progression on UV-Damaged DNA Templates in Vertebrate Cells
(A–C) Viability of wild-type (WT) and DT40 knockout cell lines including PrimPol-deficient cells expressing human PrimPol protein (PrimPol−/− + hPrimPol) was determined following exposure to UV-C (A), 4-nitroquinoline 1-oxide (4NQO; 48 hr treatment; B), and X-rays (C). Cells recovered for 48 hr after treatment before measurement of metabolic capacity. Error bars denote SD of three experiments, with two PrimPol−/− cell lines used.
(D) Alkaline sucrose sedimentation analysis of DNA from cells that were either mock or UV-C irradiated (4 J/m2) and immediately pulse-chased with 3H-thymidine. Representative of at least three experiments shown; red arrow indicates postreplication repair defect.
(E) DNA fiber analysis of cells UV-C irradiated (20 J/m2) between the CldU and IdU labeling periods. CldU:IdU ratio distribution representative of two sets of experiments using two PrimPol−/− cell lines (Cl1 and Cl2) is shown; > 100 DNA fibers scored for each. The average of these data is presented as a cumulative percentage of forks at each ratio (F). See Figure S3 for details on the knockout cells.