Fluorescence resonance energy transfer (FRET) assay

For evaluation of DNA synapsis formation, oligonucleotides labeled with different fluorescence probes (Cy3 and Cy5) were annealed as described in the figure legends. Titration experiments were performed using equimolar concentration of labeled DNA (50 nM) with increasing concentrations of AtPolIs. Variations in fluorescence intensity upon incubation of AtPolIs were measured in an Infinite M1000 microplate reader using wavelengths (λ) of 530 nm for excitation and 675 nm for emission. Reactions were incubated in 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1.5 mM DTT, 10% glycerol, 0.1 mg/ml BSA and varying concentrations of AtPoIls. The reactions were incubated for 20 min in darkness at 25°C by triplicate. The data were normalized and analyzed in GraphPad Prism 6.