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	Figure 1. FANCD2 recruits BLMcx to replicating chromatin independently of FANCI. (A) FANCD2, FANCI and BLMcx members exhibit overlapping chromatin-binding patterns in S-phase. Sperm chromatin was replicated in Xenopus S-phase extracts and re-isolated at the indicated time points. Chromatin fractions (lanes 2–8) were analyzed for bound FANCD2, FANCI and BLMcx members. Lane 1: 1 µl extract (loading control). Inset: replication assay. Replication was monitored by pulsing replicating extract aliquots with [α-32P]dGTP at the indicated time windows. (B) Immunodepletion of FANCD2 or FANCI from S-phase extracts does not co-deplete BLMcx proteins. S-phase extracts were mock-, FANCD2 or FANCI-depleted and analyzed for the presence of FANCD2, FANCI and BLMcx members. (C) Recombinant FANCD2WT restores BLMcx recruitment to replicating chromatin in FANCD2/FANCI double-depleted extracts. S-phase extracts were mock depleted (lanes 1 and 2), FANCD2/FANCI depleted (lanes 3 and 4) or FANCD2/FANCI depleted and reconstituted with myc–FANCD2WT (lanes 5 and 6). Sperm chromatin was allowed to replicate in the different extracts, isolated at the indicated time points and analyzed for bound FANCD2, FANCI and BLMcx members. (D) Recombinant FANCD2WT—but not FANCD2K562R—restores BLMcx recruitment to replicating chromatin in FANCD2-depleted extracts. S-phase extracts were mock depleted (lanes 1 and 2), FANCD2 depleted (lanes 3 and 4) or FANCD2 depleted and reconstituted with either myc–FANCD2WT (lanes 5 and 6) or myc-FANCD2K562R (lanes 7 and 8). Sperm chromatin was replicated in the different extracts, re-isolated at indicated time points and analyzed for bound FANCD2, FANCI and BLMcx members. Inset: FANCD2-depleted extracts fail to promote monoubiquitination of supplemented myc-FANCD2WT (owing to low residual FANCI levels) but support chromatin recruitment of non-ubiquitinated myc-FANCD2WT and myc-FANCD2K562R. Chromatin fractions isolated from mock-depleted (lane 1) or FANCD2-depleted (lane 2) extracts, or from FANCD2-depleted extracts supplemented with myc-FANCD2WT (lane 3) or myc-FANCD2K562R (lane 4) were run on a low-percentage gel to distinguish non-ubiquitinated from monoubiquitinated FANCD2 isoforms.
	Figure 2. Defective BLMcx chromatin recruitment in FANCD2-depleted extracts is not caused by replication delay. (A) FANCD2-depleted S-phase extracts exhibit a delay of replication onset. S-phase extracts were mock depleted (lanes 1–4) or FANCD2 depleted (lanes 5–8). Sperm chromatin was added to extracts and replication was monitored by pulsing replicating extract aliquots with [α-32P]dGTP at the indicated time windows. (B) FANCD2—but not FANCI—is responsible for timely replication onset. S-phase extracts were mock depleted (lanes 7 and 8), FANCD2/FANCI depleted (lanes 1 and 2) or FANCD2/FANCI depleted and supplemented with myc-FANCD2WT (lanes 3 and 4) or Flag-FANCIWT (lanes 5 and 6). Sperm chromatin was replicated in the different extracts, and replication was monitored by pulsing extract aliquots with [α-32P]dGTP at the indicated time windows. (C) Parallel to the replication assay shown in Figure 2A, replicating chromatin was re-isolated at indicated time points from mock-depleted extracts (lanes 1–5) or FANCD2-depleted extracts (lanes 6–9), and chromatin fractions were analyzed for bound FANCD2, FANCI and BLMcx members.
	Figure 3. FANCD2 is required for full BLM complex assembly. (A) Input panels: S-phase extracts were mock depleted (lanes 1 and 2) or FANCD2 depleted (lanes 3 and 4), and either untreated (lanes 1 and 3) or incubated with dsDNA fragments for 10 min (lanes 2 and 4). (B) IP panels: the differently depleted egg extracts described in (A) were subjected to IP with rabbit IgG (lanes 1 and 4) or a Xenopus-specific BLM antibody (lanes 2, 3, 5 and 6). Input and IP samples were analyzed for the presence of FANCD2, FANCI and BLMcx proteins.
	Figure 4. FANCD2 protects BLM protein stability. (A) FANCD2 protects BLM protein stability in replicating S-phase extracts. Egg extracts were mock depleted (lanes 1–4), FANCD2 depleted (lanes 5–8) or FANCD2 depleted and treated with MG132 (lanes 9–12). Chromatin was replicated in extracts, and chromatin-containing extract aliquots were taken at indicated time points and analyzed for FANCD2, FANCI and BLMcx members. (B) BLM stabilization does not restore BLMcx chromatin binding in FANCD2-depleted extracts. From replicating extracts described in (A), chromatin fractions were isolated at indicated time points and analyzed for chromatin-bound FANCD2, FANCI and BLMcx. (C) myc-FANCD2WT restores BLM protein stability in FANCD2-depleted extracts. Egg extracts were mock depleted (lanes 1, 4 and 7), FANCD2 depleted (lanes 2, 5 and 8) or FANCD2 depleted and supplemented with myc-FANCD2WT (lanes 3, 6 and 9). Chromatin was replicated in the extracts and chromatin-containing extract aliquots were taken at indicated time points and analyzed for FANCD2, FANCI and BLMcx members. (D) FANCI is dispensable for BLM protein stability. Egg extracts were mock depleted (lanes 1–4), FANCI depleted (lanes 5–8) or FANCI depleted and treated with MG132 (lanes 9–12). Chromatin was replicated in the extracts, and chromatin-containing extract aliquots were taken at indicated time points and analyzed for FANCD2, FANCI and BLMcx members.
	Figure 5. FANCD2 regulates DNA DSB-triggered hyperphosphorylation of BLM and RPA2 independently of FANCI. S-phase egg extracts were mock depleted (lanes 1–3), FANCD2 depleted (lanes 4–6) or FANCI depleted (lanes 7–9), and supplemented with 50 ng/μl dsDNA fragments. Extract aliquots were taken at the indicated time points and analyzed for FANCD2, FANCI, BLM and RPA2.
	Figure 6. FANCD2-dependent BLM regulation is conserved in human cells. (A) Human BLM forms a constitutive complex with non-ubiquitinated FANCD2. Left panel: WCE were prepared from PD20 + D2 cells (lanes 1 and 2) and subjected to IP with rabbit IgG (lane 3), FANCD2 antibody (lanes 4 and 5) or BLM antibody (lanes 6 and 7). Input and equal volumes of IP samples were analyzed for presence of FANCD2 and BLM. Right panel: The same samples shown in the left panel were reanalyzed by extended gel electrophoresis. Sample volumes were adjusted to contain equal amounts of FANCD2 to better discern between non-ubiquitinated and monoubiquitinated FANCD2 isoforms. N.B.: to achieve equal FANCD2 protein concentrations in WCE and IP lanes, 1/30 of the original sample volumes were loaded in lanes 1, 2, 4 and 5. (B) Left panel: Human FANCD2 supports BLM protein stability. WCE were prepared from human PD20 + D2 cells (lanes 1 and 2) and PD20 cells (lanes 3 and 4) that had been untreated (lanes 1 and 3) or treated with 30 µM APH (lanes 2 and 4) and analyzed for FANCD2 and BLMcx members. Middle panel: Human FANCD2 mediates BLMcx chromatin recruitment. PD20 + D2 cells (lanes 1 and 3) and PD20 cells (lanes 2 and 4) were untreated (lanes 1 and 2) or treated with 30 µM APH for 3 h (lanes 3 and 4). Chromatin fractions were isolated from the cells and analyzed for presence of FANCD2, BLM and TOP3a. Histone H3: loading control. Right panel: Human FANCD2 is crucial for BLMcx assembly. WCE from untreated or APH-treated PD20 + D2 and PD20 cells were subjected to IP with rabbit IgG (lanes 1 and 4) or BLM antibody (lanes 2, 3, 5 and 6), and IP samples were analyzed for the presence of FANCD2 and BLMcx members.
	Figure 7. Human FANCD2 and BLM act in one pathway to mediate replication fork restart and suppression of new origin firing. (A) Human cell types used for DNA fiber analysis: wild type (PD20 + D2, siControl), FANCD2 deficient (PD20, siControl), BLM deficient (PD20 + D2, siBLM) and FANCD2/BLM double deficient (PD20, siBLM). (B) Images of DNA fibers with a schematic of defining sites of replication. Red tracts: DigU; green tracts: BioU. (C) FANCD2 and BLM act in a common pathway to mediate replication fork restart after replication blockade. The efficiency of replication restart in wild-type, FANCD2-deficient, BLM-deficient and FANCD2/BLM double-deficient cells was measured as the number of restarted replication forks after APH-mediated fork stalling (DigU→BioU tracts), compared with the total number of DigU-labeled tracts (DigU plus DigU→BioU). (D) FANCD2 and BLM act in concert to suppress new origin firing during replication blockade. The number of new sites of replication originating during the 40 min recovery period after APH treatment was compared between wild-type, FANCD2-deficient, BLM-deficient and FANCD2/BLM double-deficient cells. New origins of replication were measured as the number of green-only (BioU) tracts per unit length. ***P < 0.0001.
	Figure 8. Human BLM acts independently of FANCD2 to maintain velocity of restarted replication forks after APH treatment. (A) BLM maintains replication fork velocity in unperturbed conditions independently of FANCD2. BioU tract length distributions were determined on DigU→BioU double-labeled DNA fibers isolated from untreated (NT) wild-type, FANCD2-deficient, BLM-deficient or FANCD2/BLM double-deficient cells. (B–D) BLM maintains replication fork velocity of restarted forks independently of FANCD2. BioU tract length distributions were determined on double-labeled DNA fibers before (NT) and after APH treatment and compared between wild-type and (B) FANCD2-deficient cells, (C) BLM-deficient cells and (D) FANCD2/BLM double-deficient cells. For A–D, median tract lengths are indicated below each panel. Insets: Cumulative distributions (top) and plotted median tract lengths (bottom).
	Figure 9. Human FANCD2 acts independently of BLM to protect replication forks from degradation. Lengths of nascent replication fork tracts indicating fork stability (labeled with DigU only) were measured before (NT) and after 6 h of APH treatment. Preformed DigU tract lengths shorten during APH-treatment in (A) FANCD2-deficient (PD20) cells compared with wild-type (PD20 + D2) cells and in (B) FANCD2/BLM double-deficient (PD20, siBLM) cells compared with wild-type (PD20 + D2) cells. (C) Preformed DigU tract lengths do not shorten during APH-treatment in BLM-deficient (PD20 + D2, siBLM). Median tract lengths are indicated below each panel. Insets: Cumulative distributions (top) and plotted median tract lengths (bottom).

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