Volume 51, Issue 5, Pages 678-690 (September 2013) FANCD2 Binds MCM Proteins and Controls Replisome Function upon Activation of S Phase Checkpoint Signaling  Gérald Lossaint, Marion Larroque, Cyril Ribeyre, Nicole Bec, Christian Larroque, Chantal Décaillet, Kerstin Gari, Angelos Constantinou  Molecular Cell  Volume 51, Issue 5, Pages 678-690 (September 2013) DOI: 10.1016/j.molcel.2013.07.023 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 51, 678-690DOI: (10.1016/j.molcel.2013.07.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Proteins Identified on Nascent DNA upon Nucleotide Depletion (A) Nascent DNA was pulse labeled with EdU for 5 min in untreated HeLa S3 cells and for 50 min in HeLa S3 cells exposed to 1 mM HU. Identified proteins are represented by the ratio of normalized spectral counts detected in HU-treated versus untreated samples and ranked in the order of increasing ratio. Each dot corresponds to one protein. See also Figure S1 and Table S1. (B) STRING network view of proteins enriched at stalled forks (http://string.embl.de, default settings). Thicker lines represent stronger associations. Among the TOP80 proteins enriched ≥2.8× at stalled forks, we selected proteins that were detected in the three replicates of iPOND EdU-HU and that were absent in control thymidine chase samples. (C) Immunoblots of the indicated proteins isolated on nascent DNA. Lane 1, 5 min of EdU; lane 2, 5 min of EdU + 2 hr thymidine chase (10 mM); lane 3, 50 min of EdU in 1 mM HU; lane 4, 50 min of EdU in 1 mM HU + 2 hr thymidine chase (10 mM). Molecular Cell 2013 51, 678-690DOI: (10.1016/j.molcel.2013.07.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 MCM Proteins Copurify with FANCD2 (A) Overview of the purification procedure. (B) M2 chromatography eluates from control (M2-FLAG-BAP fusion protein) and FANCD2 purification (M2- FLAGFANCD2) were analyzed by SDS PAGE and silver staining. (C) Interaction network of replisome proteins that associate with FLAGFANCD2. For each protein identified, the number of spectral counts (SpC) is indicated. See also Table S2. (D) Western blot analysis of MCM proteins in FLAGFANCD2 pull-downs performed with or without FLAG peptide (100 μg/ml) as indicated. (E) Left, Superdex 200 fractionation of chromatin extracts from HU-treated HEK 293T cells. The positions and stokes radii of thyroglobulin (85 Å) and ferritin (61 Å) are indicated. Fractions containing FANCD2 and MCM proteins were pooled, supplemented with ethidium bromide (200 μg/ml) and DNase I (100 U/ml), and used for reciprocal coimmunoprecipitations (right). Anti-MCM3, anti-MCM5, and anti-FANCD2 immune precipitates were probed for the indicated proteins by western blotting. (F) Superose 6 fractionation of chromatin extracts from untreated (upper panel) and HU-treated (lower panel) HEK 293T cells. Molecular Cell 2013 51, 678-690DOI: (10.1016/j.molcel.2013.07.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Analysis of FANCD2 Association with MCM Proteins (A) Upper panel, chromatin extracts from FLAGFANCD2-HEK 293T cells were prepared after increasing exposure time in HU (1 mM). Lower panel, the presence of MCM3, MCM5, MCM7, and Ser108 pMCM2 proteins in FLAGFANCD2 immune precipitates was probed by western blotting. The association of FANCD2 and MCM proteins is transient and maximal after 1 hr in HU. Control immunoprecipitation (Ctrl-IP) was performed in the presence of FLAG peptide (100 μg/ml). (B) Immunofluorescence analysis of FANCD2 (green) and Ser108 pMCM2 on (red) in HDFs treated for 1 hr with 5 mM HU. DNA (Hoechst). The merge picture is an overlay of the red and green channels. A histogram indicates the percentage scored from more than 150 untreated and HU-treated cells with ≥3 colocalizing foci. Data from three independent experiments are represented as mean ± SEM. See also Figure S2. (C) When indicated, FLAGFANCD2-HEK 293T cells were pretreated for 15 min with the ATR inhibitor (ETP-46464, 1 μM) and incubated for 1 hr in 5mM HU, and chromatin extracts were prepared and probed for the indicated proteins. Ser108 pMCM2 and MCM5 in FLAGFANCD2 pull-downs with M2 agarose were probed by western blotting. (D) When indicated, FLAGFANCD2-HEK 293T cells were pretreated with Chk1 inhibitor (SB218078, 50 nM) and processed as described in (C). (E) FLAGK561R-FANCD2-HEK 293T cells were prepared and analyzed as described in (A). (F) When indicated, PD220 (FA-A) and PD220-corrected cells were cultured for 1 hr in HU (5 mM) before the preparation of chromatin extracts and immunoprecipitation of endogenous FANCD2. Ser108 pMCM2 and MCM5 were probed in the anti-FANCD2 immune precipitates. (G) FLAGFANCD2 was coexpressed with each one of the six WILD-TYPE MCM proteins in insect cells with recombinant baculoviruses. Immunoprecipitations were performed in order to verify the direct interaction of FANCD2 with MCM proteins. M2 agarose pull-downs of FLAGFANCD2 were probed for MCM proteins by western blotting. Molecular Cell 2013 51, 678-690DOI: (10.1016/j.molcel.2013.07.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 FANCD2 Controls Replisome Function upon Nucleotide Depletion (A and B) HDFs were synchronized by serum starvation for 24 hr, released in presence of serum, and labeled in S phase for 20 min with IdU and then for 2 hr with CldU in the presence of 5 mM (A) or 0.5 mM (B) HU. Graphic representations of the length of CldU tracks adjacent to IdU tracts measured in μm (y axis). The bar dissecting the data points represents the median of tract length. Differences between distributions were assessed with the Mann-Whitney rank sum test. ∗∗∗p < 0.0001. (C) A histogram showing the bin ratios of the shorter versus the longer replication track for each pair of sister replication forks labeled with CldU during exposure to HU. (D and E) HDFs were treated as described above (A and B) and washed and maintained in HU-containing medium for 16 hr in the absence of halogenated nucleotides. Next, DNA fibers were analyzed as described above: 5 mM HU (D) and 0.5 mM HU (E). See also Figure S3 and Table S3. Molecular Cell 2013 51, 678-690DOI: (10.1016/j.molcel.2013.07.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Cells Lacking FANCD2 Accumulate Replication-Associated Lesions HDFs treated with control or FANCD2 siRNAs were synchronized in S phase and then exposed for 1 hr to 5 mM HU when indicated. (A) Representative western blots showing that Ser4 and Ser8 pRPA32 and Ser345 pChk1 accumulate as a result of Chk1 inhibition (UCN01, 250 nM) or FANCD2 depletion. See also Figure S4. (B) Representatives image of HDFs treated with the indicated siRNAs, synchronized in S phase by release from serum starvation, and cultured for 1 hr in the presence of 5 mM HU prior to fixation and immunofluorescence staining of Ser4 and Ser8 pRPA32. (C) Quantification of Ser4 and Ser8 pRPA32-positive cells (expressed as a percentage) scored for more than 200 cells. Data from three independent experiments are represented as mean ± SEM. (D) Genomic DNA from HDFs treated with the indicated siRNAs and cultured for 2 hr in the presence of the indicated concentration of HU was slot blotted and probed for ssDNA with anti-guanosine. Total DNA was revealed after denaturation with the same antibody. (E) Quantification of the ratio between ssDNA and total DNA with ImageJ software. Data from three independent experiments are represented as mean ± SEM. Molecular Cell 2013 51, 678-690DOI: (10.1016/j.molcel.2013.07.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 FANCD2-Depleted Cells Exhibit Cell Proliferation Defects Due to Premature Entry into Senescence (A) Overview of the experimental procedure. HDFs were synchronized by serum starvation for 24 hr and released in presence of serum. After 12 hr, 5 mM HU was added to the cell culture medium to block the cell cycle at the G1/S transition. (B) Microphotographs (from video microscopy sequences) showing control and FANCD2-depleted HDFs immediately and 3 days after release from G1/S, as indicated. Red asterisks indicate mitotic cells. White arrows indicate cells with a senescent-like morphology. (C) A representative graph showing the number of cells in mitosis as a function of time after release from G1/S. Mitotic cells were counted from three different wells. (D) The cells were stained for SA-β-Gal activity 2 weeks after release from HU. (E) A histogram showing the percentage of cells with SA-β-Gal activity. Data from three independent experiments are represented as mean ± SEM. (F) Representative images of synchronized HDFs exposed to HU (5 mM) for 5 hr and cultured for 16 hr before fixation and p21 staining by indirect immunofluorescence. DNA was stained with Hoechst. (G) Quantification of the percentage of p21-positive cells in a population of more than 200 cells. Data from three independent experiments are represented as mean ± SEM. (H) Growth of synchronized HDFs treated with the indicated siRNAs. When indicated, S phase cells were treated with HU (5 mM) for 5 hr, washed, seeded in six-well plates (12 × 104), and the increase in cell number was determined by counting Trypan blue-excluding cells with a hemocytometer at daily intervals. Data from one representative experiment performed in triplicate are represented as mean ± SEM. Molecular Cell 2013 51, 678-690DOI: (10.1016/j.molcel.2013.07.023) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 FANCD2 Functions in Replisome Surveillance DNA synthesis is controlled by ATR signaling, which phosphorylates multiple replisome components, including MCM proteins. FANCD2 binds the MCM2–MCM7 replicative helicase and is necessary for slowing down replisomes ongoing under stressful conditions, thereby limiting the formation of replication-associated DNA damage. In cells lacking FANCD2, unrestrained replisomes produce ssDNA gaps that can be further processed by promiscuous nucleases, leading to genomic instability, p21 induction, and cell-cycle arrest. Molecular Cell 2013 51, 678-690DOI: (10.1016/j.molcel.2013.07.023) Copyright © 2013 Elsevier Inc. Terms and Conditions