Abstract
The recruitment of FANCM, a conserved DNA translocase and key component of several DNA repair protein complexes, to replication forks stalled by DNA interstrand crosslinks (ICLs) is a step upstream of the Fanconi anemia (FA) repair and replication traverse pathways of ICLs. However, detection of the FANCM recruitment has been technically challenging so that its mechanism remains exclusive. Here, we successfully observed recruitment of FANCM at stalled forks using a newly developed protocol. We report that the FANCM recruitment depends upon its intrinsic DNA translocase activity, and its DNA-binding partner FAAP24. Moreover, it is dependent on the replication checkpoint kinase, ATR; but is independent of the FA core and FANCD2–FANCI complexes, two essential components of the FA pathway, indicating that the FANCM recruitment occurs downstream of ATR but upstream of the FA pathway. Interestingly, the recruitment of FANCM requires its direct interaction with Bloom syndrome complex composed of BLM helicase, Topoisomerase 3α, RMI1 and RMI2; as well as the helicase activity of BLM. We further show that the FANCM–BLM complex interaction is critical for replication stress-induced FANCM hyperphosphorylation, for normal activation of the FA pathway in response to ICLs, and for efficient traverse of ICLs by the replication machinery. Epistasis studies demonstrate that FANCM and BLM work in the same pathway to promote replication traverse of ICLs. We conclude that FANCM and BLM complex work together at stalled forks to promote both FA repair and replication traverse pathways of ICLs.
Highlights
Bloom syndrome (BS) and Fanconi anemia (FA) are two rare genetic diseases sharing several features, suchBS is caused by mutations in BLM gene, which belongs to the RecQ DNA helicase family conserved from Escherichia coli to humans [4]
When cells are treated with DNA-damaging drugs or under replication stress, many DNA repair proteins, including BLM and FANCD2, are re-distributed to the DNA damage sites or stalled replication forks, where they can be detected as large bright foci in the nuclei [12,13,14, 45]
To detect FANCM foci under regular drug-treated conditions, we generated an antibody against chicken FANCM, Cell Discovery | www.nature.com/celldisc and found that this antibody readily detected FANCM in the bright nuclear foci in chicken DT40 cells treated with the drugs that induce replication stress, but not in the untreated cells (Figure 1a and b), or in FANCM−/ − cells treated with the same drugs
Summary
Bloom syndrome (BS) and Fanconi anemia (FA) are two rare genetic diseases sharing several features, such. BS is caused by mutations in BLM gene, which belongs to the RecQ DNA helicase family conserved from Escherichia coli to humans [4]. In addition to BLM, two other human RecQ helicases are mutated in the genomic instability diseases, Werner. The four components of this complex work coordinately to catalyze dissolution of double Holliday junctions, which are intermediates produced during the repair of DNA double-strand breaks. This leads to suppression of crossover recombination and SCEs [11]. BLM is recruited to stalled replication forks and is required for efficient recovery of the stalled forks [12,13,14,15]
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