Figure 2. Fft3 promotes DNA end resection at arrested forks through its ATPase activity.

(A) Diagram of the t-ura4sd20<ori construct, on chromosome 3, containing a single RTS1-RFB (< and blue bars) blocking replication forks travelling in the main replication direction. Main replication origins (ori, black circles) located upstream and downstream of the RTS1-RFB are indicated with their respective distances from the RFB. When Rtf1 is expressed, >90% of forks emanating from the strong centromere-proximal replication origin, and moving towards the telomere, are blocked. HR-mediated fork restart is initiated through the generation of a 1-kb-sized ssDNA gap in an Exo1-dependent manner. (B) Scheme of replication intermediates (RIs) observed in a neutral–neutral 2DGE analysis of the AseI restriction fragment upon activation of the RTS1-RFB. (C) Representative RI analysis by 2DGE in the absence (RFB OFF) or presence of fork blockade (RFB ON) in indicated strains. A DNA fragment corresponding to ura4 gene was used as probe. For each strain, numbers indicate the efficiency of the RFB; values are means of at least three independent experiments ± SD. (D) Quantification of % of fork undergoing resection (tail signals) relative to the number of blocked forks. Values are means of at least three independent experiments ± the 95% confidence interval (99% CI). In panels (C) and (D), the strains used were YC13 (WT), YC281 (fft3Δ), YC287 (fft2Δ), YC309 (fft3-myc), and YC313 (fft3-K418R-myc). (E) Binding of RPA (Ssb3-YFP) to the RTS1-RFB in indicated strains. ChIP-qPCR results are presented as an ON/OFF ratio for each strain. Upstream and downstream distances from the RFB are indicated in bp. Values are mean of four independent experiments ± SEM. P values were calculated using unpaired t test (***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05). Data from fft3Δ (KK854 strain) were compared with wild-type (KK851 strain) (blue stars) and data from fft3-K418R-myc (KK857 strain) were compared with fft3-myc strain (KK860 strain) (purple starts).