Figure 2.

The DDX21 Dimer Binds Two R-loop Molecules

(A) Mass photometric profiles obtained for DDX21_Fl (in blue) and DDX21_Fl monomer mutant (in gray) with the determined average molecular mass indicated above each peak. The theoretical molar mass for DDX21_Fl (with uncleaved affinity tag) is 104.9 kDa and for the DDX21_Fl monomer mutant is 90 kDa. The wild-type protein shows a monomer-dimer equilibrium, whereas the mutant is mainly monomeric.

(B) Mass photometric profiles for DDX21_ΔN, DDX21_Core-DD, and DDX21_core indicate that DDX21_ΔN displays a monomer-dimer equilibrium as seen for DDX21_Fl, whereas DDX21_Core-DD and DDX21_core are mainly dimeric and monomeric, respectively.

(C) Mass photometric profile for the DDX21_Fl-R-loop complex at 30 nM. (D) Fluorescence polarization binding assays measure a binding affinity of DDX21_Fl to the R-loop substrate of 9.2 ± 0.7 nM. Error bars represent the standard deviation of three independent measurements. The mass photometric experiments were performed in duplicates for the protein samples and in triplicates for the R-loop complex (see also Figure S6).