The replication and recombination intermediates on the locus at 60, 120, 180, and 240 min after release into HU were visualized by two-dimensional gel electrophoresis, followed by Southern blot analysis. contrast, the full disruption of Mms21 is lethal (19, 20, 25). The Nse1, Nse3, and Nse4 components form a trimeric subcomplex at the head and adjacent region of Smc5 (26). Little is known about the Nse5 and Nse6 subunits, except, like the other components, they are essential in genes were cloned into a pEG202-derived bait plasmid (29), creating Nse5-LexA fusion proteins under the control of a galactose-inducible promoter. was cloned into pJG4-6-derived prey vectors (29), creating a B42-activating domain fusion protein under the control of a galactose-inducible promoter. Inserting a stop codon after amino acid 96 created the Smt3GG mutant. All constructs were confirmed by sequencing, and protein expression was confirmed by Western blot analysis with anti-LexA (2-12) and anti-HA (F7) antibodies (Santa Cruz Biotechnology). Detection of Sumoylated Proteins Nickel-nitrilotriacetic acid (Ni-NTA) purification of His8-Smt3 was performed as described by Wohlschlegel (30) with the following changes. Pellets of 2 109 cells were treated with tiling arrays from Affymetrix? at the Bioinformatics and Expression Analysis Core Facility of Karolinska Institutet. Analysis and map making were performed as described previously (36). Complete maps are included in supplemental Data Sets S1CS4. Two-hybrid Analysis Constructs were transformed into JC1280. For drop assays, strains were Armillarisin A grown in the absence of glucose and plated on Armillarisin A medium containing 2% galactose and lacking His and Trp (to select for plasmids) and additionally Leu (to measure expression from reporter plasmid pSH18034. Protein-protein interactions were detected by quantitative -galactosidase activity for permeabilized cells and represent the averages of three independent experiments, with error Armillarisin A bars indicating S.D. (37). Co-immunoprecipitation Assays Cells containing HA-tagged Nse6 and Myc-tagged Smc5 were grown at 25 C to log phase before cells were lysed with glass beads in lysis buffer (50 mm HEPES, 140 mm NaCl, 1 mm EDTA, and 1% Triton X-100). Protein extracts were applied to anti-Myc antibody-coupled Dynabeads (Invitrogen) and immunoprecipitated for KDM4A antibody 2 h at 4 C. Following immunoprecipitation, samples were split and washed by shaking at 1400 rpm for 5 min once in lysis buffer and twice in wash buffer (100 mm Tris (pH 8), 0.5% Nonidet P-40, 1 mm EDTA, and either 300 mm or 1 m NaCl). Beads were resuspended in SDS loading buffer and run on 8% SDS-polyacrylamide gels, followed by Western blotting with anti-HA (F7) and anti-Myc (9E10) antibodies. RESULTS Nse5 Interacts with SUMO and Is Required for Smc5 Sumoylation We sought to characterize Nse5 within the Smc5/6 complex by utilizing two temperature-sensitive alleles, and and indicating the unmodified form), suggesting that Nse5 is not a target of sumoylation. Furthermore, Nse5 interacted with a mutant form of SUMO that cannot be conjugated to target proteins, Smt3GG (Fig. 1and and serving as a negative control (32C34). We determined the association of DNA polymerase ? by monitoring Myc-Pol2 recovery with stalled forks when cells were released into S phase in the presence of HU at the indicated time points. Compared with the wild type, we observed a reduction in polymerase association at both early-firing origins in and and and late-firing origin as described previously (32, 34). Phenotypic Analysis of nse5-ts and mms21-11 Cells The loss of viability of and the E3 SUMO ligase double mutants were not more sensitive to HU than the single mutants alone (supplemental Fig. S2double mutants grew slowly on rich medium and showed synergistic sensitivity to HU (Fig. 3and supplemental Fig. S2mutants (Fig. 3cells accumulate X-shaped DNA structures during HU treatment. (JC1908), and (JC1320) cells at 25 C. mutant cells. Smc5 sumoylation was analyzed as described for Fig. 1in WT (JC1157), (JC1155), and (JC1124) cells. (JC1718), and (JC1804) cells. (JC1908), and (JC1320) cells that were.
