Quantifying SSB-DNA Interactions by Fluorescence Anisotropy

For RPA/EcSSB/Gp32 DNA-binding, serial 2-log dilutions of the respective SSB were prepared in 30 mM HEPES pH 7.5, 30 mM NaCl, 0.25 mM EDTA, 10% glycerol, 0.01% NP-40, 1 mM DTT. Proteins were then mixed with 6-FAM-labeled oligo-dT30 (IDT) to a final concentration of 2.5 nM supplemented with 0.5 M NaCl (54 (link)). The protein–DNA mix was incubated for at least 30 min at room temperature to reach equilibrium (55 (link)). Three technical replicates were plated in a black 384-well nonbinding microplate (Greiner Bio-One) and read in a SpectraMax ID5 plate reader (Molecular Dynamics). Anisotropy values from the technical replicates were averaged and corrected by subtracting the value from a buffer control that contained no protein. Values from three independent experiments were plotted in GraphPad PRISM and fit to the Hill equation. For Pol-α/primase recruitment experiments, 6-FAM-labeled oligodT60 was pre-incubated with 2.5 nM of the respective RPA protein in the absence of supplemental NaCl. Then, Pol-α/primase was titrated into RPA-bound DNA mixture and incubated for 30 min before reading samples as described above. All samples were corrected for by subtracting a control well with no added Pol-α/primase.