Figure 3

RNR expression allows replication resumption in rad53Δexo1Δ mutants after fork stalling.

(a) Cultures of RAD53⁺, rad53ΔGAL-RNR1 and rad53Δexo1ΔGAL-RNR1 strains were grown in YPRAF medium, arrested in G1 with α-factor and released from the G1 block into RAF-containing medium containing 0.2 M HU. After 3 h in HU, cultures were released into fresh medium in the presence of Glc (left panel) or Gal (right panel). DNA content was measured by flow cytometry at the indicated times. The expression of Rnr1 was followed by immunoblot analysis (bottom panel) as described in Methods. A Ponceau-S-stained membrane coincident with Rnr1 migration was used as a loading control. Full-length blots are presented in Supplementary figure S4. (b) Analysis of S phase progression of rad53ΔEXO1⁺GAL-RNR3-2-4 and rad53Δexo1ΔGAL-RNR3-2-4 strains as described in (a). The expression of Rnr3 was followed by immunoblot analysis (bottom panel) as described in (a). (c) Analysis of S phase progression of rad53ΔEXO1⁺GAL-RNR1-2-4 and rad53Δexo1ΔGAL-RNR1-2-4 strains as described in (a). (d) rad53Δexo1ΔGAL-RNR3-2-4 cells were grown in YPRAF medium and arrested in G1 with α-factor. The culture was divided in two and incubated in either YPD (white column) or YPRAF (black column) in the presence of 0.2 M HU for 3 h. The YPD-culture was then released from HU into fresh medium with Glc, while the YPRAF-culture was released in the presence of Gal. Cells were tested for viability after α−factor arrest, after 3 h in the presence of 0.2 M HU and 2 h after release from HU. (e) Analysis of S phase progression of rad53ΔEXO1⁺dif1ΔGAL-RNR3-2-4 and rad53Δexo1Δdif1ΔGAL-RNR3-2-4 strains as described in (a).