Fig. 5: Oxidised DNA2 loses its ability to bind DNA, while maintaining a stably bound FeS cluster.

a Representative EMSA of DNA2/DNA complexes upon pre-incubation with increasing amounts of H₂O₂. DTT was added after the H₂O₂ treatment. The DNA-binding reaction was set up after H₂O₂ and DTT treatment. Final protein concentration is 50 nM and final 5′ flap DNA substrate concentration is 4 nM. Quantification of DNA binding expressed in %. Asterisk indicates addition of DTT (n = 3 independent experiments; error bars, SD). b Representative EMSA of DNA2/DNA complexes where the DNA-binding reaction was incubated for 30 min before the addition of increasing amounts of H₂O₂. Final protein concentration is 50 nM and final 5′ flap DNA substrate concentration is 4 nM. Quantification as in a (n = 3 independent experiments; error bars, SD). c Representative EMSA of DNA2/DNA complexes upon pre-incubation with H₂O₂ using 50 nM of wild-type DNA2 and 100 nM of FeS cluster-deficient variants. DTT was added after the oxidative treatment. The DNA-binding reaction was set up after H₂O₂ and DTT treatment. Quantification as in a (n = 3 independent experiments; error bars, SD). d Representative EMSA of DNA2/DNA complexes after 20 h incubation without reducing agents. DTT was added after the incubation and prior to the DNA-binding assay. Quantification as in a (n = 3 independent experiments; error bars, SD). e Representative EMSA of mDna2/DNA complexes after 20 h incubation without reducing agent. TCEP was added after the incubation and prior to the DNA-binding assay. Quantification as in a (n = 3 independent experiments; error bars, SD). f UV–vis spectroscopy with mDna2 at 25 μM, as-purified (orange), after 20 h of air exposure (teal), and after 20 h of air exposure followed by TCEP treatment (grey). The complete spectra are found in Supplementary Fig. 4f.