Figure 9

Polθ-helicase overexpression impairs the firing of replication origins. Control (WT) and HA-Polθ-helicase cells were maintained in the presence of IdU (red) and then in the presence of CldU (green). Molecules were stretched onto slides and incubated with anti-DNA (blue) to assess the molecule integrity. Because the fork direction moves from red to green, the fork direction, termination regions and origin regions in molecules can be visualized. (A) Patterns found after DNA combing. In molecules (A,B), only the direction of the fork is observable. Molecules (C–E) exhibit replication origins, and molecules (F,G) are termination zones. The arrow indicates the fork direction. *Shows patterns considered to be replication origins. (B) The percentage of molecules containing replication origins was determined. Values are the presented as the median and standard deviation of two independent experiments. Fifty molecules were analyzed in each assay. Statistical analyses were performed using Student’s t-test, *Indicates p < 0.05. (C) The graph shows the fork speed of each analyzed molecule. The length of the green fragment in kb (between the red and blue signals) was divided by the time of the green pulse in min. Statistical analyses were performed using Student’s t-test, **Indicates p < 0.01.