Disruption and characterization of FANCM helicase and nuclease domain deficient DT40 strains. (A) Map of the FANCM locus containing the exons encoding the helicase domain. The gene disruption strategy removes three exons coding for the conserved helicase domain. Genomic Southern blot confirms disruption of the helicase domain. Cartoon depicting predicted products generated by hypomorphic alleles in FANCM deficient cell lines (LMS—Large Middle Section). (B) Time course experiment of FANCD2 monoubiquitination in FANCM deficient cell lines. Cells were exposed to 5 µM cisplatin for up to 6 h. The ratios for D2-L (monoubiquitinated FANCD2) over D2-S (unmodified FANCD2) are shown for 0-h and 6-h time point. FANCD2 monoubiquitination activation in FANCM-Δhel and ΔFANCM cell lines is deficient, but normal in FANCM-Δnuc deficient cell line. FANCD2 monoubiquitination in ΔFANCL cell line is completely abolished even after 6 h of treatment. (C) Graph showing cisplatin sensitivity determined by cell viability (MTS assay) of FANCM cell lines. (D) Spontaneous SCE frequencies determined for FANCC and FANCM knockout cell lines. The elevated SCEs in the ΔFANCM strain are additive compared to ΔFANCC in the ΔFANCMΔFANCC mutant.

Sensitivity of ΔFANCM cell line to UV and camptothecin. (A) UV sensitivity curve of ΔFANCM cell line. (B) Complementation of UV light sensitivity shown by the ΔFANCM cell line harbouring +HEL but not +NUC construct. (C) Camptothecin sensitivity curve of ΔFANCM cell line. (D) CPT sensitivity of ΔFANCM cell line is partially complemented by +NUC but not +HEL construct.

FANCM functions with Blm to suppress a subset of SCE events. (A) FANCD2 monoubiquitination in the ΔBlm FANCM-D203A mutant cell line. Shown below are the ratios of FANCD2-L over FANCD2-S and fold induction before (−) and after 6 h treatment (+) with 1 µM cisplatin. (B) Cisplatin sensitivity curve as determined by cell viability (MTS assay). (C) Spontaneous SCE levels in the ΔBlm FANCM-D203A strain resemble those in the ΔBlm strain. (D) Gel filtration analisys of FANCM and FANCG in ΔBlm and ΔFANCL strains. Above—Anti FANCM immunoblot of nuclear extract fractions obtained from gel filtration. FANCM fractionates as a large molecular mass peak that does not change in ΔBlm but shifts in ΔFANCL strains. Bottom—The same fractions were also blotted with an anti-FANCG antibody to monitor the FA core complex. (E) FANCM modification after DNA damage in FA deficient cell lines. FANCM becomes modified in the absence of a functional FA core complex.

Complementation of ΔFANCM deficient cell line with FANCM domain specific cDNA. (A) The cartoon depicts the three cDNA constructs used for over-expressing three different FANCM domains in the ΔFANCM cell line. (B) Suppression by the helicase and nuclease domains of FANCD2 monoubiquitination defects observed in ΔFANCM cell line. (−) and (+) indicates untreated and treated cells exposed to 5 µM cisplatin for 17 h. Shown below is the ratio of FANCD2-L over FANCD2-S. Induction fold was calculated dividing FANCD2 ratio after by the ratio before treatment. (C) Graph showing cisplatin sensitivity as determined by cell viability (MTS assay) for all the strains shown. (D) Pull down of FANCC-TAP from nuclear extracts. Anti-FLAG immunoblot for detecting expression of FANCM domains. FANCC-TAP was detected by anti-PAP immunoblot. Left—Nuclear extract, right—FANCC-TAP immunoprecipitation (25). (E) Cellular fractionation of ΔFANCM cell lines expressing the three different FANCM domains. Above—Accumulation of FANCC-TAP into the chromatin fractions in the absence (−) or presence (+) of DNA damage. TAP blot to detect FANCC-TAP, middle—Histone H3 blot, and below—FLAG blot to detect FLAG tagged FANCM domains. (F) Spontaneous SCEs frequencies in the ΔFANCM cell line expressing the different FANCM domains.

A Walker B point mutation in the helicase domain separates the role for FANCM in SCEs suppression from that in crosslink repair. (A) FANCM immunoblot of nuclear extracts without (−) or with (+) DNA damage. A full length FANCM protein is expressed in the knock in strain that is phosphorylated following DNA damage. Below—the FANCD2 monoubiquitination process after DNA damage is not defective in FANCM-D203A cell line. (B) Cisplatin sensitivity curve as determined by cell viability (MTS assay). The strain expressing the knockin point mutation FANCM-D203A is not sensitive to cisplatin. (C) Gel filtration analysis of endogenously modified FANCM cell lines. Fractions were blotted for FANCC-TAP to detect the FA core complex. ΔFANCM and FANCM-Δhel knockout mutants but not FANCM-D203A show a smaller FA core complex size compared to wild-type. (D) High levels of spontaneous SCEs in FANCM-D203A mutant strain compared to wild-type.

Model for the role of FANCM in the DNA damage response. (A) Model for the role of FANCM in the FA pathway and crosslink repair in the DNA damage response. (1) The helicase or nuclease domain can target the FA core complex to a replication block at a DNA crosslink. (2) The core complex can stimulate FANCD2 monoubiquitination, perhaps by being in close proximity to its substrates FANCD2 and FANCI. (3) An ATP driven conformational change may enable the FANCM complex to recruit additional DNA repair proteins that carry out crosslink repair. (B) A cartoon of the FANCM protein divided into the three domains systematically dissected in this article. Each of the domains confers distinct functions to FANCM in the DNA damage response. (C) FANCM helicase suppresses crossovers by dissolving D-loops, by acting early to suppress D loop formation FANCM may prevent the formation of Holliday junction, which are then resolved by crossover.