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. 2015 Jan 20;5:7886. doi: 10.1038/srep07886

Figure 4. New DNA synthesis in rad53Δexo1ΔGAL-RNR2-3-4 mutants after replication blocks is due to fork restart.

Figure 4

(a) The experiment was performed as in Fig. 3a, with slight modifications described in (b). DNA content of rad53Δexo1ΔGAL-RNR2-3-4 cells was determined by flow cytometry at the indicated time points. (b) Replication fork progression was analysed in the rad53Δexo1ΔGAL-RNR2-3-4 strain by density transfer. Cells were initially grown in 0.1% Glc in medium with heavy isotopes, and then blocked in G1 with α−factor. After G1 arrest, cells were released into medium containing HU and light isotopes, under conditions that prevented GAL1,10-dependent expression. Cells were held in HU for 3 h and then released from the HU arrest into fresh medium with either Glc or Gal. The position of the four ClaI/SalI restriction fragments from chromosome VI that were analysed is shown at the top, coincident with ARS607 and 20, 40, and 65 kb away from the origin. (c) Percentage of replicated DNA in the rad53Δexo1ΔGAL-RNR2-3-4 strain after HU-release in the presence of GLC or GAL (from Fig. 3b). The amount of replicated DNA at each fragment was plotted against distance from the ARS607 sequence. White bars represent RNR-repressing conditions (GLC) and black bars RNR-expressing conditions (GAL).