Figure 6: MHF–FANCM-F complex possesses two DNA-binding sites.
From: The structure of the FANCM–MHF complex reveals physical features for functional assembly
(a) Calculated electrostatic on the surface of (MHF1–MHF2)2 bound to FANCM-F. Although some of the labelled residues shown in spheres (right one) having missing atoms in the final model, intact residues rebuilt in COOT were used for surface potential calculation. As a control, the surface potential of (MHF1–MHF2)2 alone are shown in (b). Red and blue surfaces represent negative and positive electrostatic potentials (−3.5 kBT, +3.5 kBT). (c) FPA of MHF1/MHF2/FANCM-F. The data was fitted according to equation (3) (Methods). Blue dashes represent the results of data fitting according to equation (2) . Clearly, it is largely divergent from the experimental data. (d) EMSA results of the FANCM-F in complex with MHF mutants. 0.6 μM DNA substrate; proteins at lanes 1–4 (MHF1K73A/R74A/MHF2/FANCM-F): 0, 5, 10, 20 μM, respectively; lanes 5–6 (MHF1/MHF2K27A/K29A/FANCM-F): 5, 20 μM, respectively; lane 7 (MHF1K73A/R74A/MHF2): 20 μM; lane 8 (MHF1/MHF2K27A/K29A): 20 μM. (e) EMSA results of the FANCM-F mutants in complex with MHF mutants. 0.6 μM DNA substrate; proteins at lanes 1–3 (MHF1R12A/R18A/K73A/R74A/MHF2/FANCM-FK686A/R690A/R693A): 0, 5, 20 μM, respectively; lanes 4–5 (MHF1K73A/R74A/MHF2/FANCM-FK686A/R690A/R693A): 5, 20 μM, respectively; lane 6-7 (MHF1K73A/R74A/MHF2R11A/K12A/FANCM-FK675A/K676A), 5, 20 μM; lane 8-9 (MHF1K73A/R74A/MHF2/FANCM-FK675A/K676A): 5, 20 μM, respectively; lane 10 (MHF1K73A/R74A/MHF2/FANCM-F): 20 μM. (f) MHF11−114/MHF2/FANCM-F still binds to DNA. But the data could only be fitted in one-site-binding mode. (g) The DNA-binding trajectory in the MHF–FANCM-F complex. MHF1 C terminus locates in the junction of the two DNA-binding paths shown in orange strip, and the MHF1 C-terminal residues may follow a trajectory similar to that of α4 helix of NC2α shown in blue.
