FANCD2 regulates BLM complex functions independently of FANCI to promote replication fork recovery

Fanconi Anemia (FA) and Bloom Syndrome share overlapping phenotypes including spontaneous chromosomal abnormalities and increased cancer predisposition. The FA protein pathway comprises an upstream core complex that mediates recruitment of two central players, FANCD2 and FANCI, to sites of stalled replication forks. Successful fork recovery depends on the Bloom’s helicase BLM that participates in a larger protein complex (‘BLMcx’) containing topoisomerase III alpha, RMI1, RMI2 and replication protein A. We show that FANCD2 is an essential regulator of BLMcx functions: it maintains BLM protein stability and is crucial for complete BLMcx assembly; moreover, it recruits BLMcx to replicating chromatin during normal S-phase and mediates phosphorylation of BLMcx members in response to DNA damage. During replication stress, FANCD2 and BLM cooperate to promote restart of stalled replication forks while suppressing firing of new replication origins. In contrast, FANCI is dispensable for FANCD2-dependent BLMcx regulation, demonstrating functional separation of FANCD2 from FANCI.


Coomassie blue staining of the polyacrylamide gel showing purified recombinant Xenopus laevis wildtype and ubiquitination dead mutant FANCD2 proteins
Chaudhury et al.

Figure S 2
Legend: S-phase extracts were mock-depleted (lanes 1 and 2), FANCI-depleted (lanes 3 and 4) or FANCI-depleted and reconstituted with myc-FANCD2 WT (lanes 5 and 6). Sperm chromatin was added to the extracts and allowed to replicate. Chromatin was reisolated at the indicated time points, and analyzed for bound FANCD2, FANCI and BLMcx members. Legend: (A) BLM, RPA2 and FANCD2 colocalize in nuclear foci during late S-phase. Sperm nuclei were replicated in Xenopus S-phase extracts and reisolated at 30 min (mid-replication) and 90 min (late replication) and analyzed for the presence of FANCD2, BLM and RPA2 by immunofluorescence analysis. N.B.: We were not able to show colocalization of FANCD2 and BLM foci since the available Xenopus-specific antibodies against both proteins were generated in rabbits.

Chaudhury et al.
FANCD2 regulates foci formation of BLM and RPA2 S33-P during late S-phase

Figure S 3 continued
(B) FANCD2 WT , but not FANCD2 K562R rescues BLM and RPA2 S33-P foci formation in FANCD2depleted extracts. Sperm nuclei were replicated in S-phase extracts that were undepleted, FANCD2-depleted, or FANCD2-depleted and supplemented with recombinant FANCD2 WT or FANCD2 K562R . Nuclei were isolated at 90 min and analyzed for foci formation of BLM and RPA2 S33-P .

(C and D)
Histogram of quantitative analysis of nuclei containing BLM (C) and RPA2 S33-P (D) foci in the differently depleted extracts described in (B). Nuclei were tabulated in groups containing 0-10 and ≥ 11 foci per nucleus.
Chaudhury et al.  Legend: PD20+D2 (wildtype control for PD20) cells and GM00637 (wildtype control for GM08505) cells were either untreated or treated with APH (30 μM) or HU (2mM) for 2, 6 or 24 hours. (A) The efficiency of replication restart was measured as the number of restarted replication forks after APH or HUmediated fork stalling (DigU→BioU tracts), compared to the total number of DigU-labeled tracts (DigU plus DigU→BioU tracts). 80% of replication forks restarted in PD20+D2 and GM00637 cells following APH or HU-mediated stalling for 2 h and 6 h, but not 24 h. (B) The new sites of replication visible during the 40 min recovery period after APH or HU treatment were measured as the number of green-only (BioU) tracts per unit length. New origin firing was suppressed in PD20+D2 and GM00637 cells after APH-or HU-mediated replication fork stalling for 2 h and 6 h, but not 24 h.

PD20+D2 and GM00637 cells exhibit normal replication restart responses after 6 h treatment with APH or HU
new sites of replication measured as the number of only green tracts per unit length   (38) and is recruited to the stalled fork. Here, FANCD2 fulfills two distinct functions: (a) it recruits BRCA2 and stabilizes RAD51 at the fork to prevent fork degradation (2) and (b) it assembles the BLM complex at the fork and mediates BLMcx phosphorylation to promote replication fork restart. Simultaneously, FANCD2 and BLM act in concert to suppress firing of new replication origins.
Chaudhury et al.