Full-length AtALBA1 and AtALBA2 CDSs were amplified and cloned into the pET28a expression vector for protein purification. The K30E mutation was introduced into the construct through site-directed mutagenesis with the QuikChange II XL Site-Directed Mutagenesis Kit according to the manufacturer’s instructions (Agilent Technologies). The proteins were expressed in Escherichia coli DE3 (BL21) cells and purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. EMSA was performed as previously described (19). Oligonucleotide sequences used in this study are described in table S1. The indicated DNA or RNA oligonucleotides were synthesized and labeled with biotin at the 5′ end. Next, the oligonucleotides were annealed to a complementary strand by heating them to 95°C for 5 min and cooling them slowly. The annealing created dsDNA, dsRNA, a DNA-RNA hybrid, and an R-loop structure. The oligonucleotides (5 nM) were incubated with AtALBA1 or AtALBA2 recombinant proteins at 25°C for 10 min in the binding buffer [20 mM tris-HCl (pH 7.6), 10 mM MgCl2, and 1 mM DTT]. The resulting protein-substrate complexes were resolved on 4% nondenaturing polyacrylamide gels at 80 V for 80 min using 1× TBE buffer (89 mM tris-HCl, 89 mM boric acid, and 2 mM disodium EDTA). After electrophoresis, the oligonucleotides in the gels were detected using a chemiluminescent biotin-labeled nucleic acid detection kit (D3308, Beyotime).

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