Structural insights into the functions of the FANCM-FAAP24 complex in DNA repair

Abstract

Fanconi anemia (FA) is a genetically heterogeneous disorder associated with deficiencies in the FA complementation group network. FA complementation group M (FANCM) and FA-associated protein 24 kDa (FAAP24) form a stable complex to anchor the FA core complex to chromatin in repairing DNA interstrand crosslinks. Here, we report the first crystal structure of the C-terminal segment of FANCM in complex with FAAP24. The C-terminal segment of FANCM and FAAP24 both consist of a nuclease domain at the N-terminus and a tandem helix-hairpin-helix (HhH)2 domain at the C-terminus. The FANCM-FAAP24 complex exhibits a similar architecture as that of ApXPF. However, the variations of several key residues and the electrostatic property at the active-site region render a catalytically inactive nuclease domain of FANCM, accounting for the lack of nuclease activity. We also show that the first HhH motif of FAAP24 is a potential binding site for DNA, which plays a critical role in targeting FANCM-FAAP24 to chromatin. These results reveal the mechanistic insights into the functions of FANCM-FAAP24 in DNA repair.

Figure 1.

Structure of the FANCM-FAAP24 complex. (A) EMSA of the DNA-binding ability of FANCM-FAAP24 on ssDNA. The reaction contained the biotin-labeled ssDNA (10 nM) without or with increasing amounts of FANCM-FAAP24 (0.0 µM, lanes a, f and k; 0.05 µM, lanes b, g and l; 0.1 µM, lanes c, h and m; 0.4 µM, lanes d, i and n; and 0.8 µM, lanes e, j and o). The asterisk represents the biotin label at the DNA 5′ end. The arrows indicate the shifted bands of the protein–DNA complexes, and the dot represents the free DNA probe. (B) Ribbon representation of the overall structure of FANCM-FAAP24. The nuclease and the (HhH)₂ domains of FANCM are colored in salmon and yellow, and those of FAAP24 in cyan and violet, respectively. The secondary structure elements are labeled with those of FAAP24 designated with apostrophes. The disordered regions are indicated with dotted lines.

Figure 2.

Interactions between FANCM and FAAP24. (A) Interactions between the nuclease and the (HhH)₂ domains of FANCM. The interface is mainly mediated by residues from α4 and the β2-β3, β3-β4, α4-β5 and α6-α7 loops of the nuclease domain, and α8 and the α7-α8 and α10-α11 loops of the (HhH)₂ domain. (B) Interactions between the nuclease domains of FANCM and FAAP24. The interface is mainly mediated by residues from α4, α5 and β6 of FANCM and α3′, α4′, β6′ and the α4′-α5′ loop of FAAP24. (C) Hydrophilic interactions and (D) hydrophobic interactions between the (HhH)₂ domains of FANCM and FAAP24. The interface is mainly mediated by residues from α7, α9 and α11 of FANCM and α5′, α6′, α7′ and α9′ of FAAP24. The nuclease and the (HhH)₂ domains of FANCM are colored in salmon and yellow, and those of FAAP24 in cyan and violet, respectively. The interacting residues are shown with ball-and-stick models. The hydrogen bonds are indicated with black dashed lines and the salt bridge with gray dashed line.

Figure 3.

Structural comparison of the FANCM-FAAP24 complex with other XPF family members. (A) Comparison of the active sites of FANCM (salmon), FAAP24 (cyan), ApXPF (PDF code 2BGW, light blue) and DrMus81 (PDB code 2ZIU, magenta). Residues corresponding to the catalytic GDX_nERKX₃D motif in FANCM, FAAP24, ApXPF and DrMus81 are shown with ball-and-stick models. Only the secondary structure elements of ApXPF are labeled. In the color coding scheme, the nuclease domains are abbreviated as N. Mg²⁺ in ApXPF-dsDNA is shown with a green sphere. (B) Electrostatic surface representations of the active sites in ApXPF-dsDNA and FANCM-FAAP24. The bound dsDNA in ApXPF-dsDNA is shown with orange ribbon and the modeled dsDNA with gray ribbon. ApXPF-dsDNA in the right panel and FANCM-FAAP24 are shown in the same orientation as these in Figure 3A. Mg²⁺ in FANCM-FAAP24 was modeled based on ApXPF-dsDNA and is shown with a green sphere.

Figure 4.

A potential DNA-binding site in FAAP24. (A) Localization of the wild-type and truncated FANCM-FAAP24. Scale bars: 10 µm. (B) Electrostatic surface (left panels) and distribution of the conserved residues on the surface (right panels) of FANCM-FAAP24. The two positively charged surface patches in the (HhH)₂ domains are indicated with orange circles. The active site of FANCM is indicated by a modeled Mg²⁺ shown with a green sphere. The color coding scheme of the residue conservation is indicated. In the color coding scheme, the nuclease and the (HhH)₂ domains are abbreviated as N and H, respectively. (C) Localization of the mutant FANCM-FAAP24. Scale bars: 10 µm. (D) EMSA of the wild-type and mutant FANCM-FAAP24. The reaction contained the biotin-labeled ssDNA, dsDNA and splayed-arm DNA (10 nM) without or with FANCM-FAAP24 (0.0 µM, lane a; 0.4 µM, lanes b, d and f; 0.8 µM, lanes c, e and g). The asterisks represent the biotin labels at the DNA 5′ end. The arrows indicate the shifted bands of the protein–DNA complexes, and the dots represent the free DNA probes.