Figure 1

EXO1 affects replication fork restart in rad53Δ cells after HU arrest.

(a) Drop assays on YPD plus the indicated concentrations of HU were performed using 1:5 serial dilutions of exponentially growing cultures of RAD53⁺EXO1⁺, rad53ΔEXO1⁺ or rad53Δexo1Δ strains. SML1 was previously deleted in all of them to allow viability in the absence of RAD53. (b) The strains were synchronized in G1 with α−factor and released into YPD medium containing 0.2 M HU for 2 h. HU was then removed and cells were released into fresh YPD medium for 2 h. Samples were taken every 30 min and DNA content was measured by flow cytometry. (c) Replication fork progression was followed by density transfer, as described in the text. Cells were blocked in G1 with α-factor and released into medium containing HU for 90 min. HU was then removed and cultures were released into HU-free medium for 120 min. DNA content at the indicated time points is shown in Supplementary Fig. S1a. The sizes and positions of the restriction fragments 1 and 3 of the analysed replicon on chromosome VI are represented in the scheme. The relative amounts of radioactivity in the hybridized DNA are plotted against the gradient fraction number. The position of unreplicated (HH) and replicated (HL) DNA peaks is indicated at the top. Black arrows indicated fork movement from ARS607. The position of the initial HH peak is shown as a reference (gray area). (d) The graph shows the percentage of replicated DNA at positions ARS607 and fragment 3 in the RAD53⁺ strain (white bars), rad53Δ strain (gray bars) and rad53Δexo1Δ strain (black bars) at the end of the experiment shown in Figure 1C.