Figure 6.

Rad51 S14 Is Important for Replication Restart after Replication Stress

(A) Western blot analysis of U2OS cell lines expressing exogenous Rad51 WT or S14A, after downregulation of endogenous Rad51 using siRNA targeting its 3′ UTR.

(B) Percentage of HU-treated (4 mM for 4 hr) Rad51 WT and S14A U2OS cells containing more than 20 γH2A.X foci. Error bars, SEM (n = 3).

(C) Illustration of the DNA fiber experimental setup, with incorporation of IdU and CldU shown in red and green, respectively. Representative IF images of DNA fibers defined as replication restart (incorporation of both IdU and CldU), stalled replication fork (RF; IdU only), and new origin firing (CldU only) are shown. The bar indicates 10 μm.

(D) Recovery of DNA synthesis was evaluated by the percentage of labeled tracts containing both IdU and CldU against the total number of labeled tracts. Error bars, SEM (n = 3).

(E) Stalled replication forks were evaluated by the percentage of labeled tracts containing only IdU. Error bars, SEM (n = 3).

(F) Firing of new origins after HU treatment was evaluated by the percentage of labeled tracts containing only CldU. Error bars, SEM (n = 3).

In panels (D)–(F), asterisks indicate unpaired two-tailed Student’s t test; ^∗∗∗p < 0.001, ^∗∗p < 0.01. See also Figure S6.