FANCM of the Fanconi anemia core complex is required for both monoubiquitination and DNA repair - PubMed (original) (raw)

. 2008 Jun 1;17(11):1641-52.

doi: 10.1093/hmg/ddn054. Epub 2008 Feb 19.

Affiliations

FANCM of the Fanconi anemia core complex is required for both monoubiquitination and DNA repair

Yutong Xue et al. Hum Mol Genet. 2008.

Abstract

In response to DNA damage, the Fanconi anemia (FA) core complex functions as a signaling machine for monoubiquitination of FANCD2 and FANCI. It remains unclear whether this complex can also participate in subsequent DNA repair. We have shown previously that the FANCM constituent of the complex contains a highly conserved helicase domain and an associated ATP-dependent DNA translocase activity. Here we show that FANCM also possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimics intermediates generated during homologous recombination or at stalled replication forks. Using an siRNA-based complementation system, we found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA-crosslinking drug, mitomycin C, but not for the monoubiquitination of FANCD2 and FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair.

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Figures

Figure 1.

Figure 1.

FANCM helicase domain has high binding affinity for 4WJ and fork DNA. (A) Coomassie-stained SDS–gel showing the baculoviral recombinant protein containing the helicase domain of FANCM (rFANCM-Hel). (B) A gel-shift assay showing DNA-binding preference of rFANCM-Hel using a variety of synthetic DNA substrates illustrated at the bottom. The p32-labeled probe is denoted with an asterisk. The arrow indicates DNA-bound rFANCM-Hel. [(C) and (D)] Gel-shift assays showing binding of rFANCM-Hel to fork (C) and 4WJ (D) at increasing protein concentrations. (E) A competition experiment showing rFANCM-Hel has higher affinity to 4WJ than fork or dsDNA. The p32-labeled 4WJ DNA was used at 0.6 n

m

, and the non-labeled competitor DNA concentration was as high as about 200-fold in excess (125 n

m

). The quantifications of (C)–(E) are shown in

Supplementary Material, Figure S2

.

Figure 2.

Figure 2.

FANCM has branch-migration (BM) activity for movable 4WJ DNA in both directions. (A) A silver-stained SDS–gel showing the Flag-tagged FANCM of either wildtype (WT) or the K117R ATPase point mutant isolated from EBNA-HEK293 cells as described previously (15). A mock purification was done using EBNA-HEK293 cells that do not express any tagged protein. (B) A BM assay showing wildtype FANCM can branch-migrate a movable 4WJ (25), but the point mutant (K117R) cannot. The 4WJ substrate (lane 3), a duplex intermediate used to assemble the 4WJ (lane 2), and the final BM product (lane 1) are illustrated on the left. This 4WJ has a 1 bp mismatch to inhibit spontaneous BM, which is illustrated as two angles in the BM product. The spontaneous BM was monitored using the DNA substrate without any added protein (none). The labeled ssDNA is marked with an asterisk. [(C) and (D)] BM assays showing that FANCM can branch-migrate partial 4WJ substrates in directions of both 5′ to 3′ (C) or 3′ to 5′ (D). The substrates have either a 5′ (C) or a 3′ (D) protruding ssDNA, and only BM in the aforementioned direction (marked by an arrow) can yield the expected BM product. A possible binding activity between FANCM and the substrates was observed (marked by an arrowhead). The unassembled duplex refers to a DNA intermediate used to assemble the partial 4WJ substrate. We were unable to fully convert these intermediates into the final substrates despite repeated efforts. Control experiments showed that these duplex intermediates should not interfere with the BM assay, because they cannot be dissociated by FANCM to produce ssDNA that has mobility similar to the BM product (data not shown).

Figure 3.

Figure 3.

The FANCM helicase domain, but not its associated ATP-dependent activity, is required for FANCD2 monoubiquitination. (A) A flow-chart shows an siRNA-based complementation system to study FANCM function in HeLa cells. (B) A graphic presentation showing the various FANCM constructs and siRNA used in the study. Notably, siRNA-O targets the open-reading frame (ORF) and should deplete both endogenous and exogenous FANCM. In contrast, siRNA-U targets the 3′-untranslated region (3′-UTR) and should deplete only the endogenous, but not the exogenous, FANCM, because this region is absent in the latter. [(C)–(F)] Immunoblotting shows the effects of FANCM depletion on FANCD2 monoubiquitination in HeLa cells (C), and HeLa-derived cell lines that stably expressing FANCM-WT (D), its helicase deletion mutant (FANCM-ΔHel) (E) and the K117R ATPase point mutant (F). The monoubiquitinated and unubiquitinated forms of FANCD2 are marked by FANCD2-L and FANCD2-S, respectively. The ratio between FANCD2-L and the total FANCD2 (both L and S) was obtained by using TotalLab100 image analysis software and shown at the bottom of each figure (L/L+S). FANCM-L and FANCM-S represent hyper- and hypo-phosphorylated forms of this protein (15). The absence or presence of MMC is indicated. A control siRNA (con) was included.

Figure 4.

Figure 4.

The ATP-dependent function of FANCM is required for cellular resistance for MMC. [(A)–(C)] Graphs showing that the MMC hypersensitivity of the HeLa cells depleted of endogenous FANCM can be complemented by exogenous wildtype FANCM (WT) (A), but not its helicase deletion mutant (FANCM-ΔHel) (B), or the K117R ATPase mutant (FANCM-K117R) (C). Cells treated with control siRNA (con), the siRNA targeting 3′-untranslated region (UTR), or the open-reading frame (ORF) of FANCM are indicated. Notably, the ORF siRNA depletes both endogenous and exogenous FANCM, whereas the UTR siRNA depletes only the former but not the latter (Fig. 3). Thus, the difference between the results of two siRNAs should reflect the effect of the exogenous FANCM. The assay was done in duplicate, and three independent assays were done for each clone. We noticed that the control-siRNA-treated HeLa cells expressing FANCM-ΔHel mutant are more resistant to MMC than those expressing full-length FANCM protein. This is likely due to reduced toxicity of FANCM protein after its helicase domain is deleted. Perhaps, expression of FANCM from the exogenous viral promoter confers certain growth disadvantage or toxicity to their hosts. This may help to explain our failure to obtain stable expression of FANCM in FANCM-deleted patient or DT40 cells.

Figure 5.

Figure 5.

The FANCM helicase domain, but not its associated ATP-dependent activity, is required for monoubiquitination of both FANCI and FANCD2. (A) Immunoblotting shows that monoubiquitination of FANCI and FANCD2 is reduced in HeLa cells depleted of FANCM by siRNA. The ubiquitinated and non-ubiquitinated forms of these two proteins are marked by L (for long) and S (for short), respectively. The long- and short-form of FANCM represent hyper- and hypo-phosphorylated version of this protein, respectively (15). The presence or absence of mytomycin C (MMC) and hydroxyurea (HU) are indicated. Notably, the induction of monoubiquitination is higher in the presence of HU than MMC (compare lane 2 with 6). The ORF siRNA was used for FANCM depletion in this experiment. A non-specific polypeptide crossreactive with FANCM antibody is indicated with an asterisk. [(B)–(E)] Immunoblotting shows the effects of FANCM depletion on FANCI and FANCD2 monoubiquitination in HeLa cells (B), and HeLa-derived cell lines that stably expressing FANCM-WT (C), its helicase deletion mutant (FANCM-ΔHel) (D) and the K117R ATPase point mutant (E). A control siRNA (con) was included. The 3′-UTR and ORF siRNA oligos are described in Figure 3.

Figure 6.

Figure 6.

FANCM can form homodimers through its C-terminal domain. (A) Graphic presentations of HA-tagged FANCM constructs used in establishing stable HeLa cell lines and co-immunoprecipitation analysis. The full-length wildtype FANCM (WT-FL), the helicase deletion mutant (ΔHel), the C-terminal deletion mutant (WT-ΔC) and the K117R ATPase point mutant in the context of C-terminal deletion (K117R-ΔC) are shown. (B) Immunoblotting analysis of the polypeptides that co-immunoprecipitate with different HA-tagged FANCM proteins as described in (A). The asterisk indicates the endogenous FANCM that co-immunoprecipitates with FANCM-ΔHel. Notably, very little endogenous FANCM was detected in the immunoprecipitate of FANCM-WT-ΔC or FANCM-K117R-ΔC mutants, suggesting that the C-terminal domain of FANCM is important for FANCM dimerization. (C) Graphs to illustrate different GST- or 6-histidine-tagged (His) FANCM and FAAP24 proteins used in the pull-down assays in (D). These bi-cistronic vectors have been used previously to show that bacterially expressed GST-FANCM can specifically associate with FAAP24 (21). (D) The GST-pull-down assay shows that GST-FANCM not only stably associates with His-FAAP24, as described previously (21), but also can associate with His-FANCM. As a control, the GST protein alone fails to pull down His-FANCM. The number of the amino acid residues in the FANCM fusion proteins is indicated as subscript, and both FANCM fusion proteins include the entire ERCC4 endonuclease and helix–hairpin–helix domains. The degradation products are marked with a bracket. We found that these degradation products have higher affinity to the glutathione beads than the GST-FANCM fusion proteins, suggesting that FANCM reduces the binding of GST to the beads.

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