Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Sep 3;21(9):1648-58.
doi: 10.1016/j.str.2013.07.006. Epub 2013 Aug 8.

Architecture and DNA recognition elements of the Fanconi anemia FANCM-FAAP24 complex

Rachel Coulthard  1 , Andrew J Deans, Paolo Swuec, Maureen Bowles, Alessandro Costa, Stephen C West, Neil Q McDonald
Affiliations

Architecture and DNA recognition elements of the Fanconi anemia FANCM-FAAP24 complex

Rachel Coulthard et al. Structure. .

Abstract

Fanconi anemia (FA) is a disorder associated with a failure in DNA repair. FANCM (defective in FA complementation group M) and its partner FAAP24 target other FA proteins to sites of DNA damage. FANCM-FAAP24 is related to XPF/MUS81 endonucleases but lacks endonucleolytic activity. We report a structure of an FANCM C-terminal fragment (FANCMCTD) bound to FAAP24 and DNA. This S-shaped structure reveals the FANCM (HhH)2 domain is buried, whereas the FAAP24 (HhH)2 domain engages DNA. We identify a second DNA contact and a metal center within the FANCM pseudo-nuclease domain and demonstrate that mutations in either region impair double-stranded DNA binding in vitro and FANCM-FAAP24 function in vivo. We show the FANCM translocase domain lies in proximity to FANCMCTD by electron microscopy and that binding fork DNA structures stimulate its ATPase activity. This suggests a tracking model for FANCM-FAAP24 until an encounter with a stalled replication fork triggers ATPase-mediated fork remodeling.

PubMed Disclaimer

Figures

Figure 1
Figure 1
FANCM-FAAP24 Complex Depicted with Electron Microscopy (A) Schematic of FANCM-FAAP24 domain structure. (B) Schematic showing the reconstruction of FANCM (orange) and FAAP24 (blue). Reference-free class averages containing characteristic views of FANCM-FAAP24 particles (raw 1, 3), or of FANCM alone (raw 2, 4). Increased contrast is used in rows 3 and 4 to aid particle comparison. Cyan arrows point at the additional density, which we assign to FAAP24. (C) Fluorescence polarization assay measurement of FANCMCTD-FAAP24 affinity for ssDNA and dsDNA. Measurements for (C) and (D) were performed in triplicate on at least three independent protein preparations. Kd values were calculated for each experiment, averaged, and an SEM was calculated. (D) Fluoresence polarization assay measurement of the FANCM1–669 DNA translocase domain’s affinity for different branched and unbranched DNA. (E) Stimulation of FANCM1–669 ATPase activity by forked DNA structures.
Figure 2
Figure 2
Crystal Structure of an S-Shaped FANCMCTD-FAAP24 Complex Bound to dsDNA (A) Schematic representation of the asymmetric unit. Calcium ions are shown as black spheres. Linked colored spheres show number and approximate location of residues for the linker connecting the FANCM-PND to its (HhH)2 and the FAAP24 NLD to its (HhH)2. Both linkers and one loop that had no electron density in the crystal structure are presumably disordered. (B) DNA footprint on dsDNA for A. pernix XPF (red) and FAAP24 (cyan) (HhH)2 domains. (C) Rotation of the (HhH)2 domains relative to the superposed ND/PND/NLD-domains from the A. pernix XPF and FANCMCTD-FAAP24 structures. Approximate location of the (HhH)2 dimer axis is shown in blue for FANCM-FAAP24, orange for A. pernix XPF. See also Figure S2.
Figure 3
Figure 3
FAAP24 (HhH)2 Domain Makes Functionally Important Contacts with dsDNA (A) Detailed view of FAAP24 (HhH)2 domain interaction with dsDNA through minor groove phosphates. (B) Targeted mutations in a FAAP24 hairpin affect the dsDNA equilibrium dissociation constants relative to a wild-type control in the fluorescence polarization assay (average of three experiments; error bars show SEM). Values shown are the quantification of the histogram. Measurements were performed in triplicate on at least three independent preparations. The ratio of equilibrium dissociation constants of the mutant protein relative to wild-type was calculated for each experiment, averaged, and an SEM was calculated. (C) Scheme of the complementation system. Endogenous FAAP24 is knocked down by transfection of STEALTH RNAi but the exogenous Flag-FAAP24 contains an siRNA-resistant wobble mutation (yellow). (D) Western blotting of FAAP24 and FANCD2. FANCD2 ubiquitinated and nonubiquitinated forms are indicated. %Ubi is the quantified ratio of ubiquitinated to nonubiquitinated FANCD2 forms. (E) Indirect immunofluorescence of cells treated with 40 ng/ml mitomycin C for 7 hr as described in the Experimental Procedures. The percentage of nuclei with > 5 foci scored from 300 nuclei counted is shown in the inset for each example. (F) Cell survival after treatment with mitomycin C was measured using the SRB assay. Points represent the average of two experiments, each performed in quadruplicate, ± average SE.
Figure 4
Figure 4
A Divalent Metal Site within the FANCMCTD Pseudo-Nuclease Domain Is Essential for FANCM Function (A) Schematic of selected side chains close to the divalent metal binding site within FANCM PND. (B) Mutations in the metal-binding cleft, but not the equivalent cleft of FAAP24, increase the equilibrium dissociation constants for dsDNA relative to the wild-type protein. Values shown are the quantification of the histogram. Measurements were performed in triplicate on at least three independent preparations. The ratio of equilibrium dissociation constants of the mutant protein relative to wild-type was calculated for each experiment, averaged, and an SEM was calculated. (C) Scheme of the complementation system. Endogenous FANCM is knocked down by the stable siRNA but the exogenous Flag-FANCM contains an siRNA-resistant wobble mutation (yellow). (D) Western blot analysis of FANCM and FANCD2. Asterisk denotes nonspecifc band detected with FANCM antibody. FANCD2 ubiquitinated and nonubiquitinated forms are indicated. %Ubi is the quantified ratio of ubiquitinated to nonubiquitinated FANCD2 forms. (E) Indirect immunofluorescence of cells treated with 40 ng/ml mitomycin C for 7 hr as described in the Experimental Procedures. The percentage of nuclei with > 5 FANCD2 or γH2AX foci scored from 300 nuclei counted is shown in the inset for each example. (F) Cell survival after treatment with mitomycin C was measured using the SRB assay. Points represent the average of three experiments, each performed in quadruplicate, ± SE. See also Figure S4.
Figure 5
Figure 5
Bipartite Duplex Recognition within FANCMCTD-FAAP24 Requires Distortion of dsDNA (A) Extending the 11 base pair dsDNA (yellow) compared to the experimentally observed dsDNA (orange) glances past the metal center of FANCM PND domain. (B) Effect of combining all four DNA-binding mutations (D4) in FANCMCTD-FAAP24 on the equilibrium dissociation constant for ss- and dsDNA normalized against wild-type protein (left). D4 refers to FANCM:E1864Q/R1866A-FAAP24:V172E/K173E. Right, electrophoretic mobility shift assay showing wild-type and D4 mutant binding to ss and dsDNA. Measurements were performed in triplicate on at least three independent preparations. The ratio of equilibrium dissociation constants of the mutant protein relative to wild-type was calculated for each experiment, averaged, and an SEM was calculated. (C) Distortion of dsDNA is required to proceed from the FAAP24 hairpins into the FANCM PND metal-binding cleft to permit simultaneous engagement of both DNA-binding regions of FANCMCTD-FAAP24. Selected residues referred to in the text are shown as sticks including K24 from FAAP24 and R1860 near a dsDNA symmetry contact (grey surface).
Figure 6
Figure 6
Model for FANCM-FAAP24 Tracking and Stimulation of ATPase Activity by Fork Encounter A cartoon representation of FANCM-FAAP24 is shown tracking along dsDNA until an encounter with a stalled replication fork. At this point, the fork stimulates FANCM ATPase activity, resulting in fork remodeling and initiation of DNA repair.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Baker N.A., Sept D., Joseph S., Holst M.J., McCammon J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA. 2001;98:10037–10041. - PMC - PubMed
    1. Berger I., Fitzgerald D.J., Richmond T.J. Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol. 2004;22:1583–1587. - PubMed
    1. Blackford A.N., Schwab R.A., Nieminuszczy J., Deans A.J., West S.C., Niedzwiedz W. The DNA translocase activity of FANCM protects stalled replication forks. Hum. Mol. Genet. 2012;21:2005–2016. - PubMed
    1. Chang J.H., Kim J.J., Choi J.M., Lee J.H., Cho Y. Crystal structure of the Mus81-Eme1 complex. Genes Dev. 2008;22:1093–1106. - PMC - PubMed
    1. Ciccia A., Ling C., Coulthard R., Yan Z., Xue Y., Meetei A.R., Laghmani H., Joenje H., McDonald N., de Winter J.P. Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM. Mol. Cell. 2007;25:331–343. - PubMed

Publication types

MeSH terms

Associated data