Phosphor screen

AGO2^−/− cells were infected with MSCV retroviral constructs to stably express FH-AGO2^WT or FH-AGO2^5XA. FH-AGO2-expressing cells were seeded using 1.5×10⁷ cells per dish in 15 cm dishes with three dishes per cell line. Lysates were generated using methods similar to the co-immunoprecipitation assays, with the exception that 2 mL of lysis buffer was used per dish. Lysates were diluted with one volume of lysis buffer. FH-AGO2 was immunoprecipitated using 9 μg of anti-FLAG antibody (F1804, Sigma) and 150 μL of washed Dynabeads. Samples were rotated at 4°C overnight. Beads were washed three times with lysis buffer and then treated with lambda protein phosphatase (NEB) for 45 minutes. Beads were washed three times with lysis buffer and then resuspended in 100 μL reaction buffer composed of 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2.5 mM DTT, 0.01% Triton X-100, 0.5 mg/mL BSA, 0.5 mM EGTA, 0.5 mM Na₃VO₄, 5 mM beta-glycerophosphate, 170 ng of recombinant CSNK1A1 (PV3850, Thermo Fisher), and 200 μM [γ-³²P]ATP (SA = 100-500 cpm/pmol). Reactions were incubated at 37°C for 2 hrs. Beads were separated and mixed with 50 μL of 2X Laemmli sample buffer. SDS-PAGE was performed, and gels were stained using SimplyBlue SafeStain (Invitrogen). ³²P signal was detected using a phosphor screen (GE Healthcare) and Typhoon FLA 7000 (GE Healthcare).

Each binding reaction contained 125 total radioactive counts with final reaction conditions of: 20 mM Tris-HCl, pH 8, 150 mM NaCl, 10% glycerol, 1 mM DTT, 0.5 μg tRNA (Ambion), 1 μg BSA (Invitrogen), and <90 pM RNA. To anneal RNA, oligos were snap-cooled by heating at 95°C for 1 min and cooled on ice for 1 hour. For capped RpL30 shifts and capped purine-substituted controls, final concentrations of 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 nM Larp-DM15 were titrated. For capped Non1 shifts, final concentrations of 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, and 1000 nM Larp-DM15 were titrated. Native 7% polyacrylamide 0.5X TBE gels were pre-run on ice at 120 V for 30 min. Binding reactions were run at 120 V on ice for 45–52 min. Gels were dried for 30 min and allowed to expose overnight using a phosphor screen (GE). Screens were imaged using GE Amersham Typhoon. Bands were quantified using ImageQuant TL (GE). Background subtraction was first done using the rolling ball method and then subtracting the signal from the zero-protein lane from each of the shifted bands. Fraction shifted was determined by dividing the background-corrected intensity of the shifted band by total intensity of bands in each lane. Three independent experiments were done for each oligo, with the average plotted and standard deviation shown.

Throughout this study, we used the kinase-dead version (K227A) of Rad53, fused to the Glutathione-S-transferase (GST) purification tag (GST-Rad53-kd), as substrate for Mec1 kinase. Phosphorylation of 600 nM Rad53, or as indicated, was performed in a 10 μl assay containing 25 mM Hepes-NaOH pH 7.4, 2 % glycerol, 1 mM DTT, 20 μg/ml BSA, 0.08 % ampholytes pH 3.5-10, 8 mM Mg-acetate, 100 μM ATP, 0.5 μCi [γ −³²P] ATP or as indicated, 40 or 100 mM NaCl final concentration (including contributions made by protein storage buffers), and 3 nM Mec1. Reactions were initiated by adding the indicated concentrations of Dpb11, Dna2-499, or Dna2-1 peptide ^{8 (link)}, and incubated at 30 °C for 10 minutes. Reactions were quenched by adding 5 μl of 2.5x SDS-PAGE loading dye and heated at 95°C. Samples were separated on 8 % SDS-PAGE gel, dried, and exposed to phosphor screen (GE healthcare) and imaged with a phosphoimager. The bands were quantified using ImageQuant and plotted using KaleidaGraph. The activity of the Mec1 mutants was evaluated with respect to wild-type Mec1, overexpressed and purified analogously. Most assays were carried out in triplicate and the error bars in the Figures are standard error of the mean (s.e.m). Assays with mutants with low or no activity were carried out in duplicate and the plots show the average without error bars.