A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability

Abstract

FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.

Figure 1. Identification of MHF1 and MHF2 as integral components of the FA core complex

(A) Immunoblotting showing Superose 6 gel-filtration profiles of FANCM, MHF1 and MHF2 in HeLa nuclear extract. The peak fractions and those corresponding to marker proteins are indicated at the bottom. (B) A silver-stained gel showing that the complex purified by a FANCM antibody from the pooled peak 1 fractions in (A) contained MHF1, MHF2 and other components of FA core and BLM complexes. IP indicates immunoprecipitation. (C) Immunoblotting shows that MHF1, MHF2 and other FA core complex components were present in the immunoprecipitates isolated from peak 1 fractions by MHF1, FANCM or FANCA antibodies. Nuclear extract (NE) was used as a loading control. (D) As described in (B), except a MHF1 antibody was used in IP. (E) Immunoblotting shows that MHF2 co-immunoprecipitated with MHF1 and other FA core complex components from HeLa cells stably expressing Flag-tagged MHF2, but not from control HeLa cells. A Flag antibody was used in IP. (F) As described in (B), except that peak 2 fractions in (A) were used for IP by a MHF1 antibody. (see also Figures S1 and S2).

Figure 2. MHF binds double-strand and branch-structured DNAs, but not single-strand DNA

(A) Coomassie blue-stained SDS-PAGE gels showing HIS-tagged recombinant proteins MHF1, MHF2 and MHF complex purified from E.coli. A nonspecific band was marked with “x”. (B) Electrophoretic mobility shift assay (EMSA) for testing DNA binding activity of MHF1, MHF2 and MHF complex. A variety of DNA substrates (0.5nM) illustrated at the top were incubated with 0.6 μM MHF1, 0.6 μM MHF2 and increasing amounts (0.3 and 0.6μM) of MHF complex, respectively. Asterisks denote ³²P label at the DNA 5’ end. The arrows indicate the shift bands of MHF-DNA complex. The dots represent free DNA probe. (see also Figure S3).

Figure 3. MHF stimulates DNA binding and fork reversal activities of FANCM

(A) A silver-stained gel showing FANCM-MHF complex immunoprecipitated directly by a MHF1 antibody from HeLa nuclear extract (NE). Three major polypeptides were identified as FANCM, MHF1 and MHF2 by mass spectrometric analyses. (B) A diagram shows the mapping of MHF-interaction domain within FANCM. Left panels show FLAG-tagged wild type and various deletion mutants of FANCM used in Figure S4D. Right panels show the presence or absence of interactions between various FANCM constructs and MHF. (C) EMSA showing the DNA binding activity of MHF and FANCM₇₅₄. The reaction contained the DNA substrate (0.5nM) with or without recombinant FANCM₇₅₄ protein or MHF complex as indicated. The protein concentrations are: FANCM₇₅₄: 22.5, 45 and 90 nM in lanes 2 to 4 and 6 to 8, respectively; MHF: 300 nM in lanes 5 to 8; FANCM₇₅₄: 15nM in lanes 10, 14, 15, 16, 18, 22, 23, 24, 26, 30, 31, 32; MHF: 50, 100 and 150nM in lanes 11 to 13, 14 to 16, 19 to 21, 22 to 24, 27 to 29, and 30 to 32, respectively. The shifted bands of indicated protein-DNA complex and free DNA probe are illustrated. (D) A silver-stained gel showing Flag-FANCM purified from baculovirs-infected insect cells (Sf21), either alone or in association with the MHF complex (left panels). At these protein concentrations, MHF proteins were hardly detectable by silver- staining due to their low molecular mass. Western blots confirmed that MHF1 and MHF2 were co-immunoprecipitated with Flag-FANCM (right panels). (E) Comparison of the DNA binding activity of purified FANCM and FANCM-MHF complex on fork DNA substrate by EMSA. 5’-³²P-labeled synthetic replication forks (0.5 nM) were incubated for 30 min at room temperature with increasing concentrations of FANCM or FANCM-MHF (0, 0.125, 0.25, 0.5, 1, 2, 4, 6, 8, 10 nM). MHF does not bind this substrate at 10nM. (F) Experimental scheme. Fork reversal converts the replication fork into a four-way junction (b). Complete fork reversal dismantles the joint molecules into nicked circular and labeled linear duplex (c). (G) Increasing concentrations of Flag-FANCM or Flag-FANCM-MHF (0, 0.125, 0.25, 0.5, 1, 2, 4, 6, 8, 10 nM) were incubated with the replication fork substrate (0.5 nM) for one hour at 37°C. The different labeled species are (top down) the four-way junction intermediate (b), the original replication fork substrate (a), and the linear duplex end product of fork reversal (c). (H) Product formation shown in (G) was quantified by PhosphorImaging and expressed as percentage of total DNA. (see also Figure S4).

Figure 4. MHF is required for stability of FANCM, activation of the FA pathway and cellular resistance to DNA damaging agents

(A and B) Immunoblotting shows that depletion of MHF1 or MHF2 reduces the level of FANCM in whole cell lysates (A) and chromatin fractions (B) of HeLa cells. Nontargeting siRNA oligos were used as a control. α-Tubulin, Histone H3 and Actin were included as loading controls. (C and D) Immunoblotting shows that HeLa cells depleted of MHF1 (C) or MHF2 (D) have reduced levels of monoubiquitinated FANCD2 and FANCI in the presence of MMC (60 ng/ml) or cisplatin (5 μM)(Cis). “L” (long) and “S” (short) represent ubiquitinated and non-ubiquitinated forms, respectively. The ratio between long and short forms was obtained by using KODAK Molecular Imaging Software and shown below the blots. (E, F and G) Clonogenic survival assays of HeLa cells depleted of MHF1, MHF2 or FANCM by siRNA following the treatment with MMC, MMS or CPT at the indicated concentrations. Three independent experiments were performed. The results were reproducible, and a representative data of mean surviving percentage with S.E.M. from triplicate cultures are shown. (see also Figure S5).

Figure 5. MHF is rapidly recruited to ICL sites in S phage cells and required for FANCM recruitment

(A) Images showing that MHF1 and FANCM were recruited to psoralen-induced ICLs beginning 15 mins after laser photoactivation in S phase cells. The yellow arrow indicates the position of the laser-targeted region. (B) Images showing that HeLa cells depleted of MHF1 or MHF2 by siRNA have deficient recruitment of FANCM to psoralen-induced ICLs. Nontargeting siRNA oligos and γ-H2AX were used as controls. Images represent 15 mins post treatment time point. The recruitment of FANCM and γ-H2AX to ICL sites was indicated by yellow and white arrows, respectively. (C) Graph showing the percentages of cells in which FANCM was recruited to psoralen-induced ICLs at various time points after photoactivation. No recruitment of FANCM to ICLs was observed in MHF2-depleted cells at 30 mins post treatment. HeLa cells were either transfected with siRNA against MHF1, MHF2 or control siRNA and synchronized in S phase prior to the experiment. Values are averages from two independent experiments with error bars representing standard error of the mean. (D) Schematic representation of the plasmid substrates used in the eCHIP assay. The presence of psoralen-ICL is indicated. Cells expressing EBNA (+) support replication of the plasmid, whereas those lacking it (-) do not. (E) Recruitment of MHF1 to the site of ICL was detected by eChIP. The enrichment of MHF1 and FAAP24 at the ICL in replicating (293EBNA) and nonreplicating (HEK293) DNA substrates was reflected by their relative recruitment ratio compared to the control substrate without ICL. The relative recruitment was derived by normalizing the comparative concentration (from real-time PCR) of crosslinked substrate to that of the unmodified control substrate. Error bars represent standard deviation from three independent experiments. (see also Figure S6).

Figure 6. MHF and FANCM act in the same pathway for FANCD2 monoubiquitination and suppression of SCE in chicken DT40 Cells

(A) Immunoblotting shows levels of FANCD2, FANCM, and MHF2 in whole cell lysates from various DT40 cells (wildtype (WT), MHF1^-/- cells, and MHF1^-/- cells complemented with human wildtype (wt) or mutant MHF1 as described in (D)). The ratio between the monoubiquitinated and unubiquitinated FANCD2 (L/S) was shown. Cells were treated with MMC (50 ng/ml) for 18 hr. BAF57 was used as a loading control. (B) Immunoblotting shows levels of monoubiquitinated and unubiquitinated FANCD2 in whole cell lysates from various DT40 cells as indicated on the top. (C) Histograms showing spontaneous SCE levels of various DT40 cells as indicated within each graph. MHF1^-/- cells transfected with human MHF1 wildtype (wt) or mutants (described in D) were also shown. The mean number of SCEs per metaphase is listed. (D) Illustration of three human MHF1 point mutants generated by substituting 2 clusters of positively-charged amino acid residues (KR and RR) to alanine as indicated. (E) A Coomassie blue-stained SDS-PAGE gel shows the purified recombinant MHF complex containing MHF1 of either wildtype (wt) or mutant A (mut A). (F) Comparison of the DNA binding activity of MHF complexes containing either wildtype or mutant A of MHF1 by EMSA. Reactions contain ³²P-labeled dsDNA substrate (0.5nM) and 0.3 or 0.6μM of the MHF complex as indicated. (G) Comparison of the DNA binding activity of FANCM₇₅₄ with either wildtype (wt) or mutant A MHF by EMSA. The reaction contained the ³²P-labeled fork DNA substrate (0.5nM) with or without FANCM₇₅₄ protein or MHF complex (wt or mut A). The protein concentrations are: FANCM₇₅₄: 22.5nM (lanes 2, 4 and 6); MHF: 450 nM (lanes 3 and 5) and 300 nM (lanes 4 and 6). (H) IP-Western analyses show association between FANCM and various Flag-tagged MHF1 point mutants in HEK293 cells. (see also Figure S7).

Figure 7. Yeast MHF and FANCM orthologs work in the same pathway to resist MMS-induced DNA damage and promote gene conversion at stalled replication forks

(A) Qualitative spot tests for strain sensitivities to MMS reveal a common role and shared pathway for Mph1, Mhf1, and Mhf2 in DNA damage resistance. (B) Quantitative analysis of MMS sensitivities shows that the triple mutants are not statistically distinguishable from srs2Δ mph1Δ at two different MMS concentrations. P values for the mhf1Δ and mhf2Δ triple mutants, respectively, versus srs2Δ mph1Δ are as follows: 0.35, 0.23 (0.01% MMS); 0.36, 0.97 (0.1% MMS). (C) Schematic showing the direct repeat recombination substrate on chromosome 3 of S. pombe plus the two types of Ade⁺ recombinants (Sun et al., 2008). (D) Ade⁺ recombinant frequencies for various strains as indicated. Error bars are the standard deviations. (E) A model describes common and unique functions of FANCM-MHF and FA core complex in signaling and repairing of blocked replication forks. The former acts in yeast, invertebrates and vertebrates, whereas the latter functions only in vertebrates. While both complexes can promote reversal of blocked forks (indicated by the red brick) to allow subsequent repair, only the FA core complex has a signaling role in monoubiquitinating FANCD2 and FANCI.