Fuji fla 9000 imager

The assay was performed essentially as described in (37 (link)). The reaction (20 μl final volume) was assembled on single primed ФX174 virion ssDNA (5 nM) in buffer O (20 mM Tris–Cl pH 7.5, 5 mM DTT, 0.1 mM EDTA, 70 mM KCl, 0.5 mM ATP, 40 μg/ml BSA, 8 mM MgCl₂, 5% glycerol and 60 μM each of dGTP and dCTP), in the presence of RPA (1 μM), PCNA (10 nM), RFC (17.5 nM), Pol δ (5 nM) followed by a 5 min incubation at 30°C to allow loading of PCNA on the substrate. DNA synthesis was initiated by adding the start buffer (60 μM dTTP and 0.375 μCi [α-32P]dATP in buffer O). After the indicated time at 30°C, the reactions were stopped with SDS (0.5% final) and Proteinase K (0.5 mg/ml) and loaded onto an agarose gel (0.8% (w/v)). After electrophoresis, the gel was dried on DE81 paper, exposed to phosphorimager screen, scanned in Fuji FLA 9000 imager and analyzed with the Multi Gauge software (Fuji).

The helicase assays with Srs2 were performed as previously described by Marini 2012 (41 (link)). Reaction mixtures containing Srs2 (10 nM) were pre-incubated with increasing concentrations of Mus81–Mms4 (12.5, 25, 50, 100, 200 and 300 nM), Mus81 (12.5, 25, 50 and 100 nM) or Mus81^1–319 (12.5, 25, 50 and 100 nM) in buffer H (30 mM Tris pH 7.5, 1 mM DTT, 0.1 mg/ml BSA, 100 mM KCl, 20 mM creatine phosphate, 20 μg/ml creatine kinase, 2.4 mM MgCl₂ and 2 mM ATP) for 10 min on ice. The reactions with Mre11 included 12.5, 25, 50, 100, 200, 300 and 400 nM of protein. Then, 3 nM of 3′ overhang DNA was added to the reactions and incubated for 10 min at 30°C. After deproteinization, by incubation with 0.1% SDS and 500 μg/ml of proteinase K at 30°C for 3 min, reactions were resolved by 12% native PAGE and the gel was scanned with a Fuji FLA 9000 imager (Fuji).