nse5-ts1 cells display replisome instability during HU-induced replication stress. A, drop assays (1:10 serial dilutions) with exponentially growing cultures were performed on YPAD ± medium containing the indicated concentrations of HU for WT (JC470), nse5-ts1 (JC1361), and nse5-ts2 (JC1833) cells at 25 °C. B–D, ChIP with anti-Myc antibody 9E10 was performed 25 °C on the following cells released from α-factor into YPAD + 0.2 m HU for Myc-Pol2: WT (JC1805), nse5-ts1 (JC1471), and nse5-ts2 (JC1914) cells. Genomic regions amplified in the ChIP analysis correspond to early-firing origins ARS305 and ARS607 and late-firing origin ARS501 as described previously (32, 34).

Formation of X-shaped molecules in nse5-ts1 mutants is Rad51-dependent. A, two-dimensional gel analysis comparing nse5-ts1 (JC1361), nse5-ts1/rad51Δ (JC2031), nse5-ts1/mms21-11 (JC1320), and nse5-ts1/mms21-11/rad51Δ (JC2030) cells that were arrested in G₁ with α-factor and released into YPAD + 0.2 m HU at 25 °C. The replication and recombination intermediates at the ARS305 locus at 60, 120, 180, and 240 min after release into HU were visualized by two-dimensional gel electrophoresis, followed by Southern blot analysis. Arrows indicate the accumulated X-shaped DNA molecules. B, drop assays (1:5 serial dilutions) with exponentially growing cultures were performed on YPAD ± medium containing 10 mm HU, and cell viability was monitored as colony outgrowth from asynchronous cultures after transient exposure to 0.2 m HU for 6 h at 25 °C, with values normalized to survival at time point 0 for WT (JC470), nse5-ts1 (JC1361), mms21-11 (JC1908), rad51Δ (JC1362), nse5-ts1/rad51Δ (JC2031), mms21-11/rad51Δ (JC2032), nse5-ts1/mms21-11 (JC1320), and nse5-ts1/mms21-11/rad51Δ (JC2030) cells.

Nse5 temperature-sensitive alleles nse5-ts1 and nse5-ts2 are unable to interact with SUMO and are defective in Smc5 sumoylation. A, drop assays on YPAD (yeast extract, peptone, adenine, and dextrose) were performed with exponentially growing cultures for which 1:10 serial dilutions were performed, and plates were incubated at 25, 30, or 37 °C to compare WT (JC470), nse5-ts1 (JC1361), and nse5-ts2 (JC1833) cells. B, sequencing of nse5-ts1 revealed four point mutations, Y111H, Y123H, N183D, and H319Y, whereas sequencing of nse5-ts2 revealed two point mutations, L70A and L247A. C and D, yeast two-hybrid analysis (described under “Experimental Procedures”) demonstrated that Nse5, but not Nse5-ts1 or Nse5-ts2, interacted with both WT Smt3 and Smt3ΔGG, a truncation that cannot be conjugated to target proteins. E, Nse5 does not appear to be a target of SUMO modification. Sumoylated proteins were purified from cells expressing endogenously HA-tagged Nse5 with (JC1960) or without (JC1355) His₈-tagged Smt3. Proteins were isolated by Ni-NTA affinity purification of His-Smt3 as described previously (30). Western blotting with anti-HA antibody failed to show a higher mobility shift band corresponding to sumoylated Nse5. F, nse5-ts1 and nse5-ts2 cells are deficient in Smc5 sumoylation. Sumoylated proteins were purified from cells expressing endogenously Myc-tagged Smc5 and His₈-tagged Smt3. Proteins were isolated by Ni-NTA affinity purification of His-Smt3 as described previously (30). Western blotting with anti-Myc antibody allowed visualization of sumoylated Smc5 in WT cells (JC1157), but not nse5-ts1 (JC1156) or nse5-ts2 (JC1884) cells or cells lacking the Smt3 tag (JC720). IP, immunoprecipitation.

nse5-ts1 and mms21-11 cells accumulate X-shaped DNA structures during HU treatment. A, drop assays (1:10 serial dilutions) with exponentially growing cultures were performed on YPAD ± medium containing the indicated concentrations of HU for WT (JC470), nse5-ts1 (JC1361), mms21-11 (JC1908), and nse5-ts1/mms21-11 (JC1320) cells at 25 °C. B, Smc5 sumoylation is further reduced in nse5-ts1 than in mms21-11 mutant cells. Smc5 sumoylation was analyzed as described for Fig. 1F in WT (JC1157), nse5-ts1 (JC1156), mms21-11 (JC1155), and nse5-ts1/mms21-11 (JC1124) cells. IP, immunoprecipitation. C–F, ChIP with anti-HA antibody (F7) was performed at 25 °C on the following cells released from α-factor into YPAD + 0.2 m HU for HA-Pol1: WT (JC1805), nse5-ts1 (JC1471), mms21-11 (JC1718), and nse5-ts1/mms21-11 (JC1804) cells. G, two-dimensional gel analysis comparing WT (JC470), nse5-ts1 (JC1361), mms21-11 (JC1908), and nse5-ts1/mms21-11 (JC1320) cells that were arrested in G₁ with α-factor and released into YPAD + 0.2 m HU at 25 °C. The replication and recombination intermediates at the ARS305 locus at 60, 120, 180, and 240 min after release into HU were visualized by two-dimensional gel electrophoresis, followed by Southern blot analysis. Arrows indicate the accumulated X-shaped DNA molecules.

Smc6 recruitment to stalled forks is reduced in nse5-ts1 mutants. BrdU/IP-chip (A) or ChIP-chip (B) with anti-FLAG antibody M2 was performed for FLAG-Smc6 in WT (CB207) and nse5-ts1 (CB1795) cells released from α-factor into 0.2 m HU for 60 min at 25 °C. The signal intensity ratio on a log 2 scale is shown on the y axis, and the chromosome coordinates are shown on the x axis for chromosomes III and X. All coordinates in the yeast genome are available in supplemental Data Sets S1–S4. C, ChIP-qPCR with anti-FLAG antibody M2 was performed for FLAG-Smc6 in WT (JC1594), nse5-ts1 (JC1665), nse5-ts2 (JC1913), and mms21-11 (JC2075) cells that were arrested in G₁ with α-factor and released into YPAD + 0.2 m HU at 25 °C as described in the legend to Fig. 2 but with more stringent wash conditions, including 300 mm NaCl and 250 mm LiCl. A probability of p = 0.001 when comparing t values of nse5-ts1 and wild type (*) indicates that the differences are statistically significant. D, shown are the results from co-immunoprecipitation of Myc-tagged Smc5 and HA-tagged Nse6 (as described under “Experimental Procedures”) with washes performed under conditions of 0.3 m NaCl or 1.0 m NaCl in WT (JC2229), nse5-ts1 (JC2230), and nse5-ts2 (JC2231) cells. IP, immunoprecipitation; ab, antibody; WCE, whole cell extract.