(A) Silver-stained gel showing the polypeptide bands purified from the nuclear extract of HT1080 cells transduced with vector alone (Mock) and cells expressing (His)₆-FLAG-tagged (HF) FANCM. The polypeptides identified by MS analysis are indicated by an arrow. (B) A silver-stained gel showing the BRAFT complex components isolated from HeLa S3 cells expressing HF-MHF1. See also Fig. S1C and S1D. (C) Immunoblot showing the presence of MHF1 and MHF2 in immunoprecipitates of core complex proteins FANCL, FANCG and FAAP100. (D) Immunoblot showing co-fractionation of FAAP24, MHF1 and MHF2 with FANCM by Superose 6 gel filtration. (E) Immunoblot showing co-purification of FA core complex components with MHF1 from EUFA867 cells transduced with vector alone (lane 2), FANCA (lane 3), FANCA and FANCM (lane 4) and FANCA and delta (C-terminus deleted) FANCM (lane 5). (F) HeLa cells were treated with MMC (100 ng/mL for 16 hours) or HU (2 mM for 16 hours) and then fractionated into S2, S3, and P, as described in Material and Methods and immunoblotted with the indicated antibodies. (G) HeLa cells were transduced with control or MHF1 shRNAs, followed by cellular fractionation into S2, S3, and P fractions. The relevant proteins were revealed by immunoblotting. (H) HeLa cells were stably transduced with control-shRNA, MHF1-shRNA or HF-MHF1, and whole-cell lysates were analyzed by immunoblotting with the indicated antibodies. Asterisk (*) denotes a nonspecific cross-reactive band. See also Fig. S1.

(A) Immunoblot showing that ablation of MHF1 expression in HeLa cells by shRNA reduces the level of monoubiquitinated FANCD2 in cells treated with MMC or HU. MHF1 knock down HeLa cells stably overexpressing HF-FANCM were used to show that phenotype observed is not due to the reduced levels of FANCM. (B) Histogram of immunofluorescence analysis of FANCD2 nuclear foci in HeLa cells in which MHF1 expression was knocked down by shRNA. Cells were either left untreated or exposed to MMC or HU, and the percentage of cells with 5 or more FANCD2 foci was determined by examining at least 150 cells. The result shows the average of 3 independent experiments with standard deviations. See also Fig. S2A. (C) & (D) Graph showing MMC and camptothecin survival curve of MHF1 knockdown cells. HeLa cells were transduced with either control or shRNA oligos targeting MHF1 and were subsequently treated with the indicated concentration of MMC and camptothecin. Visible colonies from 200 cells were counted after 10 days. The data represent the percent survival, as compared with untreated cells. Each experiment was performed in triplicate, and mean values are shown with standard deviations. HeLa cells stably expressing shRNA resistant HF-MHF1 were used as a control to show that the phenotype observed is not due to the off-target effect of shRNA. Also, MHF1 knock down HeLa cells stably overexpressing HF-FANCM were used to show that phenotype observed is not due to the reduced levels of FANCM. (E) Chromosomal aberrations following MMC treatment. HeLa cells were transfected with siRNAs and cells were treated with MMC. Metaphase spreads were prepared and scored for chromosomal aberrations. See also Fig. S2E.

(A) Recombinant MHF1-(His)₆-MHF2, MBP-MHF1, and MBP-MHF2 were expressed in and purified from E. coli. Purified proteins were analyzed by SDS-PAGE followed by Coomassie blue staining. (B) MHF1-(His)₆-MHF2, MBP-MHF1 (1.0 and 2.0 μM), and MBP-MHF2 (1.0 and 2.0 μM) were examined for their DNA binding attribute with a mixture of the four ³²P-labeled DNA substrates. (C). The indicated combinations of DNA substrates were incubated with MHF1-(His)₆-MHF2 (50, 85, 120, 155, 190, 225, 270 and 305 nM). Quantification of the data is presented in the right panels. Note that treatment of nucleoprotein complexes with SDS and proteinase K (SDS/PK) released the DNA substrates. See also Fig. S3.

(A) Amylose beads pull-down assay with FANCM and MBP-MHF1 or MBP-MHF2. S, W, and E represent supernatant, wash and elution lanes. The asterisk (*) denotes a proteolytic product of FANCM. (B) Stimulation of the branch migration activity of FANCM by the MHF1-MHF2 dimer. ATP was present in all the reactions except the one in lane 8. NP - no protein control; BM, branch migration. (C) Quantification of the results shown in figure 4B. (D) DNA branch migration by FANCM, FANCM_K117R, or the combination of FANCM or FANCM_K117R and MHF1-MHF2 was examined with or without ATP, ATP-γ-S, or AMP-PNP, as indicated. BM, branch migration. (E) ATP hydrolysis by FANCM, MHF1-MHF2 or FANCM_K117R was examined with or without ssDNA, as indicated. (F) Effect of MHF1-MHF2 on the ssDNA-dependent ATPase activity of FANCM. See also Fig. S3 and S4.