1. Volume 32, Issue 3, Pages 313-324 (November 2008)
FANCM and FAAP24 Function in ATR-Mediated Checkpoint Signaling Independently of the Fanconi Anemia Core Complex 
Spencer J. Collis, Alberto Ciccia, Andrew J. Deans, Zuzana Hořejší, Julie S. Martin, Sarah L. Maslen, J. Mark Skehel, Stephen J. Elledge, Stephen C. West, Simon J. Boulton 
Molecular Cell 
Volume 32, Issue 3, Pages (November 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Identification of FANCM and FAAP24 as HCLK2-Interacting Proteins
(A) (Left panel) Western blot showing FLAG-tagged HCLK2 expressing stable cell lines derived from the parental control line 293 Flip-In, and diagram describing preparation of cellular extracts used for purification of FLAG-tagged HCLK2. (Right panel) SYPRO Ruby-stained gel showing proteins that copurify with HCLK2 from the stable cell lines. Protein bands present in the two stable clones, but not in the control Flip-In cell line, were isolated for MS analyses. The positions of FANCM, HCLK2, and FAAP24 are highlighted on the gel.
(B) Western blots demonstrating that FANCM and FAAP24 coelute with HCLK2 specifically in the two independently derived FLAG-tagged HCLK2 expressing stable cell lines. Other known components of the FA core complex, such as FANCC, FANCB, and FANCE (data not shown), were not present in purified HCLK2 complexes. ATR, a known HCLK2-interacting protein, was used as a positive control. Arrows indicate positions of the respective protein highlighted on the left.
(C) Western blots of purified FLAG-tagged HCLK2 complexes from transiently transfected 293T cells showing that both endogenous FANCM and FAAP24 copurify with FLAG-HCLK2. ATRIP, a known HCLK2-interacting protein, was used as a positive control.
(D) Western blots of purified FLAG-tagged FAAP24 from transiently transfected 293T cells showing coimmunoprecipitation of endogenous FANCM and HCLK2.
(E) Direct interaction between HCLK2 and FAAP24 is observed in U2OS cells 24 hr after transient transfection with Myc-HCLK2 and FLAG-FAAP24 constructs. Lane 2 shows that Myc-HCLK2 is not purified with anti-FLAG beads.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2
Endogenous HCLK2, FANCM, and FAAP24 Copurify Independently of the FA Core Complex
(A) Immunoprecipitation of endogenous HCLK2 from HeLa cells, untreated or treated with 3 mM HU for 2 hr. Western blots on purified HCLK2 complexes show that endogenous FANCM and FAAP24 copurify with HCLK2 (arrow indicates the correct band for FANCM).
(B) Immunoprecipitation of endogenous FANCM with two independently derived antibodies from untreated or 3 mM HU-treated HeLa cells. Western blots on purified FANCM complexes show that endogenous HCLK2 and FAAP24 copurify with FANCM (arrow indicates the correct band for FAAP24; the nonspecific band above FAAP24 is antibody light chain).
(C) Immunoprecipitation of endogenous FAAP24 from untreated or 3 mM HU-treated HeLa cells. Western blots on purified FAAP24 complexes show that endogenous HCLK2 and FANCM copurify with FAAP24 (arrows indicate the correct bands for FANCM and FAAP24).
(D) Western blots of FANCM (left panel) and FAAP24 (right panel) complexes immunoprecipitated from nuclear extracts made from lymphoblasts of normal individuals (WT) and patients with FA due to mutations in FANCA, FANCC, or FANCG. HCLK2 coimmunoprecipitated with two independent FANCM and FAAP24 antibodies from all cell types.
(E) Size fractionation of untreated and HU-treated HeLa nuclear extracts by gel filtration. Shown are western blots of selected fractions probed for the indicated proteins. FANCM (the lower band indicated by the arrow) and FAAP24 coelute in fractions 12–18 with the checkpoint signaling factors HCLK2, ATR, and ATRIP. HU (3 mM) was added 2 hr prior to preparation of nuclear extracts.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3
FANCM and FAAP24 Depletion Leads to Increased S Phase DNA Damage in Unperturbed Cells
(A) Representative western blots of HeLa cell extracts 48 hr after siRNA treatment for the indicated protein. Arrows show the correct protein band. Actin levels are shown for each lane as a loading control.
(B) RPA foci in HeLa cells 48 hr following transfection with the indicated siRNA. Cells were either untreated or incubated with 10 μg/ml roscovitine 4 hr prior to fixation. (Left panel) Representative images of RPA foci. (Right panel) Quantification of percentage cells exhibiting RPA foci counted from over 200 cells per experiment. Bars represent mean values from three independent experiments with their associated standard errors. Scale bars represent 10 μm.
(C) γH2AX foci in HeLa cells following indicated siRNA treatments. Left panel shows representative images of γH2AX foci. Cells were either untreated or incubated with 10 μg/ml roscovitine 4 hr prior to fixation. (Right panel) Quantification of percentage cells exhibiting γH2AX foci counted from more than 200 cells per experiment. Bars represent mean values from three independent experiments with their associated standard errors. Scale bars represent 10 μm.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4
FANCM- and FAAP24-Depleted Cells Exhibit a Defective G2-M Checkpoint
(A) Quantification of nuclear abnormalities observed 48 hr after siRNA knockdown of HCLK2, FANCM, or FAAP24. (Upper panel) Mean percentage of cells exhibiting nuclear abnormities obtained from three independent experiments ± standard errors. (Lower panel) Representative DAPI-stained images of types of nuclear abnormalities observed and quantified in HCLK2-, FANCM-, and FAAP24-depleted cells. Scale bars represent 10 μm.
(B) Cdc25A degradation in HCLK2-, FANCM-, FAAP24-, Chk1-, and FANCC-depleted cells or in cells treated with the Chk1 inhibitor UCN-01. Shown are representative western blots for Cdc25A and Actin (loading control). Cells were incubated with 3 mM hydroxyurea for the indicated time in the presence 25 μg/ml cyclohexamide, after which time the cells were lysed and protein extracts made. UCN-01 (0.3 μM) was added 1 hr prior to the addition of cyclohexamide and hydroxyurea. (Lower panel) Shown are quantified levels of Cdc25A from three independent experiments for the indicated siRNA treatments. Shown are the mean values ± standard error.
(C) Assessment of G2-M checkpoint in control and HCLK2-, FANCM-, and FAAP24-depleted HeLa cells. Cells were stained for phosphorylated histone H3 (Ser10) 8 hr after exposure to 5 Gy IR (100 ng/ml nocodazole was added 1 hr after radiation exposure and cells incubated for 7 hr), and the percentage of positively staining cells was quantified by FACS analysis. Shown is the fold increase in mitotic cells in HCLK2-, FANCM-, and FAAP24-depleted cells compared to control siRNA-treated cells. Data shown are the mean from at least five independent experiments with their respective errors.
(D) Quantification of supernumery (>2) centrosomes. Cells were treated with the indicated siRNA and fixed 48 hr later. Cells were stained for γ-tubulin and scored for the presence of >2 centrosomes as described previously (Alderton et al., 2004; Griffith et al., 2008). One hundred fifty cells were analyzed per experiment, and bars represent mean values from three independent experiments ± error. ATR siRNA was used as a positive control for the SNC phenotype.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
FANCM- and FAAP24-Depleted Cells Exhibit Defective ATR/Chk1 Signaling in Response to DNA Damage
Representative images of fixed cells subjected to the indicated siRNA and then labeled by indirect immunofluorescence with the following: (A) α-Chk1 (phospho Ser317) before and 2 hr after 0.05 mM aphidicolin and 0.1 mM hydroxyurea treatment; (B) α-p53 (phospho-Ser15) before and 2 hr after 20J/m2 UV treatment; (C) α-53BP1 before and 2 hr after 3 mM HU treatment; UCN-01 (0.3 μM) was added to cells 1 hr prior to addition of HU; (D) α-FANCE (phospho-Thr346) before and 2 hr after 3 mM HU treatment. Scale bars represent 5 μm. Quantification is expressed as a percentage positive nuclear staining scored from more than 200 cells in (A) and (B) or percentage foci positive nuclei for (C) and (D). Bars represent mean from three independent experiments ± standard error.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

7. Figure 6
The DNA Translocase Activity of FANCM Is Required for Its Role in the Replication Stress Checkpoint
(A) (Upper panel) Schematic representation of full-length FANCM. Grey boxes show the DEAH helicase and mutated ERCC4 nuclease domains present in FANCM. Black bars at the extreme C terminus represent helix-hairpin-helix motifs. The K117R point mutation, which abolishes ATPase-dependent DNA translocase activity, is shown. (Lower panel) Representative western blots for αFANCM and αFLAG in extracts derived from nontargeted shRNA or FANCM-targeted shRNA HEK293 cells. Expression of WT or K117R FLAG-FANCM was achieved with shRNA-resistant cDNAs as indicated. The arrow indicates FANCM, and ∗ is a nonspecific band.
(B) Representative western blots for Cdc25A and actin (loading control) in extracts derived from nontargeting shRNAmir or FANCM-targeted shRNAmir HEK293 cells. The relative Cdc25A levels were calculated as described in Figure 4B and are indicated at the bottom of the figure.
(C) Assessment of the G2-M checkpoint in nontargeting siRNAmir, FANCM siRNAmir, and WT or K117R-corrected HEK293 cells. Experiments were carried out as described in Figure 4C. Shown is the fold increase in mitotic cells in FANCM shRNAmir-depleted and WT or K117R-corrected cells compared to nontargeting siRNA shRNAmir-expressing cells. Data shown are the mean from at least two independent experiments with their respective errors.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

8. Figure 7
Model Highlighting the Dual Role of FANCM and FAAP24 in Activation of ATR/Chk1 Signaling and Subsequent Recruitment of FA Core Complex to Repair/Resolve DNA Lesions
(A) The replication machinery (represented by the large arrows) stalls upon encountering a DNA lesion (red bar), leading to the production of regions of ssDNA. ssDNA is rapidly coated with RPA, which acts to recruit ATR-ATRIP-HCLK2. Through its ability to recognize/bind and potentially remodel stalled replication fork structures and by its ability to directly interact with HCLK2 and Chk1, FANCM and FAAP24 cooperate with ATR-ATRIP-HCLK2 and Chk1 in the sensing of replication stress and/or function as a mediator to aid in the efficient activation of the replication stress response pathway.
(B) In addition, FANCM and FAAP24 provide a landing pad to facilitate the recruitment of the FA core complex to sites of replication stress. Once recruited, the FA core complex catalyzes the monoubiquitylation of the FANCD2-FANCI heterodimer, which is essential for lesion repair. The interactions mediated by FANCM and FAAP24 link the DNA-replication checkpoint with the FA core complex, which may facilitate regulatory modifications of FA core components by ATR/Chk1 important for FA pathway activation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions