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, 7 (12), 1973-81

Fanconi Anemia Proteins Stabilize Replication Forks

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Fanconi Anemia Proteins Stabilize Replication Forks

Lily Chien Wang et al. DNA Repair (Amst).

Abstract

Fanconi anemia (FA) is a recessive genetic disorder characterized by hypersensitivity to crosslinking agents that has been attributed to defects in DNA repair and/or replication. FANCD2 and the FA core complex bind to chromatin during DNA replication; however, the role of FA proteins during replication is unknown. Using Xenopus cell-free extracts, we show that FANCL depletion results in defective DNA replication restart following treatment with camptothecin, a drug that results in DSBs during DNA replication. This defect is more pronounced following treatment with mitomycin C, presumably because of an additional role of the FA pathway in DNA crosslink repair. Moreover, we show that chromatin binding of FA core complex proteins during DNA replication follows origin assembly and origin firing and is dependent on the binding of RPA to ssDNA while FANCD2 additionally requires ATR, consistent with FA proteins acting at replication forks. Together, our data suggest that FA proteins play a role in replication restart at collapsed replication forks.

Figures

Figure 1
Figure 1. FA chromatin binding follows origin firing and precedes polymerase loading
(A) Chromatin binding of FA proteins. Cytosolic extracts were incubated with buffer, 50 ng/µL Geminin, 500 µM Roscovitine, 20 ng/µL p21Cip1, or 100 ng/µL APH, as indicated, for 10 min. Sperm nuclei were incubated with the extracts for 60 min and chromatin-bound proteins were isolated and analyzed by SDS-PAGE and Western blotting with xFANCD2, xFANCA, xFANCG, xFANCF, xFANCL, xMCM6 and RPA p70 antibodies. Bottom panel: The extent of replication in these extracts was assessed by 32P-αdCTP incorporation into genomic DNA. (B) Sperm nuclei were incubated with mock- and Pol α-depleted extracts for 60 min and chromatin-bound proteins were isolated and analyzed. Bottom panel: Soluble Pol α was monitored by Western blot in mock- and Polα-depleted extracts. (C) Sperm nuclei were incubated with mock- or RPA-depleted cytosolic extracts for 60 min and chromatin-bound proteins were isolated and analyzed. Bottom panel: Mock- and RPA-depleted extracts were probed with RPA p70 antibody. (D) Sperm nuclei were incubated with cytosolic extracts for the times indicated in the presence of 100 ng/µL APH and 5 mM caffeine, and chromatin-bound proteins were isolated and analyzed. (E) Sperm nuclei were incubated in mock- or ATR-depleted cytosolic extracts (lanes 1 and 2) or in cytosolic extract supplemented with buffer or 5 mM caffeine (lanes 3 and 4) for 60 min. Chromatin-bound proteins were isolated and analyzed. Bottom panel: Mock- and ATR-depleted extracts were probed with ATR antibody. Where shown, quantification of bands was performed with ImageJ and normalized to values in mock (B and E left panel) or buffer-treated (E right panel) values.
Figure 2
Figure 2. FANCL functions in MMC-induced lesion repair and replication restart
(A) Scheme of the experimental design. Sperm nuclei are incubated with cytosolic extract for 45 min to allow DNA replication to start, shown as replication bubbles (right). At 45 min, a DNA damaging agent (300 µM MMC, 1 ng/µL APH, or 56 µM CPT) is added with 5 mM caffeine in the presence or absence of 500 µM roscovitine. The extent of replication restart is assessed by incorporation of 32P-αdCTP into genomic DNA of samples (dashed areas represent replication restart). (B) Sperm nuclei were incubated with mock- and FANCL-depleted extracts for 60 min and chromatin-bound proteins were isolated and analyzed. (C) Mock-, FANCL- and FANCA-depleted cytosolic extracts were analyzed by SDS-PAGE and Western blotting. (D) Sperm nuclei were incubated in mock- and FANCL-depleted extracts as indicated in the scheme in 3A. 300µM MMC was added and replication restart was monitored by 32P-αdCTP incorporation for the times indicated. Chromatin was isolated and analyzed by agarose gel. (E) Lanes from the gel in 3D were quantified using a PhosphorImager. CR: Caffeine and Roscovitine, C: Caffeine
Figure 3
Figure 3. FANCL promotes the restart of collapsed but not stalled replication forks
(A) Sperm nuclei were incubated in mock- or FANCL-depleted cytosolic extracts. 300 µM MMC (black bars), 40 µM APH (white bars), or 56 µM CPT (gray bars) were used in the replication restart, as indicated, with the addition of 5 mM caffeine, 32P-αdCTP, and with or without 500 µM Roscovitine (+ or −) for 90 min. Chromatin was isolated and analyzed by agarose gel and quantified as described above. Values from FANCL-depleted experiments were expressed as a percentage of the corresponding mock-depleted values. Each bar represents the average of at least 3 experiments – mean +/− SEM. * Statistically significant with p-value = 0.0007 ** Statistically significant with p-value = 0.0015 (B) As in 3A, with mock- or FANCD2-depleted cytosolic extracts. * Statistically significant with p-value = 0.0378 ** Statistically significant with p-value = 0.0123 (C) As in 3A, with extracts supplemented with buffer or 50 µM curcumin. * Statistically significant with p-value = 0.0011 ** Statistically significant with p-value = 0.0126

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