Fig. 4: Replication forks are unstable in the absence of MCM8/9 and degraded by a combination of nucleases.
a Cells were labeled with CldU followed by IdU for the indicated time intervals followed by 2 mM HU for 4 h. Experiments containing plasmids for GFP-MCM8, GFP-MCM9, or GFP alone (Ø) were transfected for 48 h prior. DNA was spread by gravity to measure NSD and representative fibers are shown. b IdU and CldU lengths for 293T WT (gray), 8KO (blue), or 9KO (red) cells were measured using ImageJ and the corresponding median ratios reported at the top of the plots and by a black line embedded in the data. Open circles (○) represent nontreated (NT) conditions, while solid closed circles (●) represent HU-treated cells. Green circle outlines (○) represent transfected cells. Cell lines were transfected with siRNA twice for 24 h each to knockdown c Mre11, d treated with 10 µM mirin, or knockdown of e Exo1 or f Dna2 and DNA fibers processed as above. siRNA knockdown was verified by western blot. Greater than 150 fibers were measured for each condition. A Mann–Whitney two-sided U test was used to calculate P values (n.s. nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
