The Fanconi Anemia Protein FANCM Can Promote Branch Migration of Holliday Junctions and Replication Forks

Molecular Cell

Short Article The Fanconi Anemia Protein FANCM Can Promote Branch Migration of Holliday Junctions and Replication Forks Kerstin Gari,1 Chantal De´caillet,1 Alicja Z. Stasiak,2 Andrzej Stasiak,2 and Angelos Constantinou1,* 1Department

of Biochemistry, University of Lausanne, Ch. des Boveresses 155, 1066 Epalinges s/Lausanne, Switzerland for Integrative Genomics, University of Lausanne, 1015 Lausanne-Dorigny, Switzerland *Correspondence: [email protected] DOI 10.1016/j.molcel.2007.11.032 2Center

SUMMARY

Fanconi anemia (FA) is a genetically heterogeneous cancer-prone disorder associated with chromosomal instability and cellular hypersensitivity to DNA crosslinking agents. The FA pathway is suspected to play a crucial role in the cellular response to DNA replication stress. At a molecular level, however, the function of most of the FA proteins is unknown. FANCM displays DNA-dependent ATPase activity and promotes the dissociation of DNA triplexes, but the physiological significance of this activity remains elusive. Here we show that purified FANCM binds to Holliday junctions and replication forks with high specificity and promotes migration of their junction point in an ATPase-dependent manner. Furthermore, we provide evidence that FANCM can dissociate large recombination intermediates, via branch migration of Holliday junctions through 2.6 kb of DNA. Our data suggest a direct role for FANCM in DNA processing, consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks. INTRODUCTION Fanconi anemia (FA) is genetically heterogeneous with 13 FA proteins having been identified to date, out of which eight proteins, including FANCM (Meetei et al., 2005), form a nuclear complex, whose integrity is required for the monoubiquitination of the FA proteins FANCD2 and FANCI in response to DNA replication stress (Garcia-Higuera et al., 2001; Sims et al., 2007; Smogorzewska et al., 2007). Monoubiquitinated FANCD2 accumulates in the nucleus within DNA repair foci containing BRCA1, RAD51, and BRCA2 (Garcia-Higuera et al., 2001; Hussain et al., 2004), the latter controlling the activity of RAD51 during DNA double-strand break repair by homologous recombination (Esashi et al., 2005; Yang et al., 2005). The discovery of biallelic BRCA2 mutations in FA complementation group D1 provides a further link between FA and DNA repair (Howlett et al., 2002). Beyond its role in FANCD2 monoubiquitination, recent evidence suggests that the FA core complex may have an additional func-

tion in DNA damage tolerance (Matsushita et al., 2005), but none of the subunits has been shown to have a direct role in DNA repair. The N-terminal part of FANCM is able to bind single-stranded DNA in vitro, albeit with low affinity (Mosedale et al., 2005). FANCM is orthologous to an archaeal protein known as Hef (Nishino et al., 2005), both sharing an N-terminal DEAH box helicase domain and a C-terminal ERCC4 nuclease domain. FANCM displays DNA-dependent ATPase activity and promotes the dissociation of DNA triplexes in vitro (Meetei et al., 2005; Mosedale et al., 2005). Recently, FAAP24 (FA-associated protein of 24 kDa) has been identified as a protein that interacts with the C-terminal region of FANCM. FAAP24 associates with the FA core complex and is required for normal levels of FANCD2 monoubiquitination (Ciccia et al., 2007). Whereas the C-terminal part of FANCM alone does not bind DNA, a complex of FAAP24 and the C terminus of FANCM binds preferentially to 30 flap and splayed arm DNAs. It has therefore been suggested that FAAP24 may recruit the FA core complex to DNA (Ciccia et al., 2007). FANCM is endowed with a helicase and an endonuclease domain and is therefore likely to bind DNA. Due to the difficulty to produce the recombinant full-length protein, DNA binding studies have been performed with the N-terminal and the C-terminal regions of FANCM, which show little and no DNA binding, respectively. Here we report on the successful purification of full-length FANCM from a recombinant source. We show that FANCM binds specifically and with high affinity to branched DNA molecules, such as Holliday junctions and replication forks. Furthermore, we provide evidence that FANCM is able to translocate the branchpoint of model DNA substrates, which mimic DNA replication and repair intermediates.

RESULTS FANCM Binds Preferentially to Branched DNA Molecules To investigate the biochemical properties of FANCM, we expressed a recombinant FANCM protein carrying a Flag tag at its N terminus in Sf21 insect cells. FANCM was purified by affinity chromatography, using anti-Flag agarose and ssDNA cellulose (Figure 1A).

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Figure 1. FANCM Binds to Branched DNA Molecules (A) Silver-stained SDS gel (6%–14% gradient) showing purified FANCM carrying an N-terminal Flag tag isolated from Sf21 insect cells (lane 2). Size marker (lane 1). (B) Autoradiographs showing the binding of FANCM to different oligo-based DNA substrates. Above each panel, a schematic representing the tested DNA substrate is shown. Asterisks indicate 32P label at the DNA 50 end. In lanes 1–5, 6–10, ., 41–45, increasing amounts of FANCM (0, 0.25, 1, 2.5, and 5 nM) were incubated with 0.5 nM of the indicated DNA substrate and the protein-DNA complexes were resolved on 6% polyacrylamide gels. (C) Autoradiographs showing the binding of 2.5 nM FANCM to 0.5 nM radiolabeled Holliday junctions (lane 2) and replication forks (lane 7). In lanes 3 and 8, FANCM was incubated with an anti-Flag antibody prior to binding to DNA. In lanes 4 and 9, anti-WHIP antibody served as a negative control. () free DNA; () FANCM-DNA complexes; and () anti-Flag antibody-FANCM-DNA complexes.

Binding of FANCM to different oligonucleotide-based DNA structures (Tables S1 and S2 available online) was investigated by electrophoretic mobility shift assays (Figure 1B). FANCM exhibited a strong affinity for branched DNA structures, such as model Holliday junctions and replication forks (Figure 1B; Figure 1C, lanes 2 and 7). To exclude the possibility that DNA was bound by a contaminating protein, we included an antiFlag antibody to the reaction mixtures to target FANCM. This led to the formation of an antibody-protein-DNA complex whose mobility was retarded further (Figure 1C, lanes 3 and 8; Figure S1). A control antibody (anti-WHIP) did not cause supershifts (Figure 1C, lanes 4 and 9), and the anti-Flag antibody did not affect the mobility of DNA molecules in the absence of FANCM (Figure 1C, lanes 5 and 10).

FANCM Binds to Holliday Junctions and Replication Forks with High Specificity and Affinity Gel retardation assays with radiolabeled Holliday junctions and replication forks carried out in the presence of nonradioactive DNA competitors underlined the high preference of FANCM for model Holliday junctions (Figure 2A) and replication forks (Figure 2B). 10–20 nM of nonradioactive Holliday junctions and replication forks were sufficient to reduce by half the amounts of FANCM complexes with radiolabeled Holliday junctions (Figure 2A) and replication forks (Figure 2B), whereas 500 nM of nonradioactive ssDNA and dsDNA molecules was necessary to produce the same effect. In order to determine the affinity of FANCM for its DNA substrates, we used saturation binding assays in which a constant

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Figure 2. FANCM Binds to Holliday Junctions and Replication Forks with High Specificity and Affinity (A and B) Graphical representation of binding of FANCM to radiolabeled Holliday junctions and replication forks in the presence of nonlabeled DNA competitors. 2.5 nM FANCM was incubated with 0.5 nM radiolabeled Holliday junctions (A) and replication forks (B), respectively, in the presence of the indicated amounts of nonlabeled DNA competitors. Binding was analyzed by gel electrophoresis and quantified by PhosphorImaging. (C and D) Measurement of the affinity of FANCM for Holliday junctions (C) and replication forks (D) by Scatchard plot analysis. A constant amount of FANCM (0.5 nM) was incubated with different amounts of radiolabeled DNA substrates, as indicated. Dissociation constants (KD) were determined by quantitative analysis of EMSAs. The ratio of bound versus free DNA substrates was plotted against the concentration of bound DNA, whereby KD = 1/slope. (E and F) FANCM (2.5 nM) and 0.5 nM radiolabeled Holliday junctions (E) and replication forks (F) were preincubated for 15 min at room temperature. An excess (10 mM) of nonlabeled Holliday junctions (E) or replication forks (F) was then added, to trap any FANCM molecules that dissociated from radiolabeled DNA. Samples were resolved by gel electrophoresis 0, 5, 10, 20, 30, and 60 min after addition of competitor DNA. Quantification of protein-DNA complexes was done by PhosphorImaging. Koff = 0.693/t1/2.

amount of protein was titrated with increasing amounts of radiolabeled DNA substrates (Figures 2C and 2D). Scatchard plot analysis (Scatchard, 1949) yielded apparent dissociation constants (KD) of 6 3 1011 M for Holliday junctions and 1.2 3 1010 M for replication forks. We next assessed the stability of FANCM-DNA complexes. To do so, FANCM was preincubated with radiolabeled Holliday junctions (Figure 2E) and replication forks (Figure 2F), respectively, before a large excess of nonlabeled Holliday junctions (Figure 2E) or replication forks (Figure 2F) was added in order to trap any FANCM molecules that dissociated from radiolabeled DNA. Time course analyses demonstrated that the half-life of protein-DNA complexes was 17.5 min for Holliday junctions (Koff = 0.040 min1), and 12.5 min for replication forks (Koff = 0.055 min1). We conclude that FANCM exhibits specific and high affinity for branched DNA molecules, such as Holliday junctions and repli-

cation forks, and that FANCM-DNA complexes show a low dissociation rate. FANCM Promotes Branch Migration of Holliday Junctions and Replication Forks In the light of its strong affinity for branched DNA structures, we investigated whether FANCM was able to promote branch migration of Holliday junctions and replication forks. We first used a Holliday junction substrate containing a central homologous core of 26 bp flanked by terminal regions of heterology (Constantinou et al., 2001). This substrate was not branch migrated to dissolution by FANCM (data not shown). This is not surprising, because FANCM is unable to separate DNA strands (Meetei et al., 2005), and the dissolution of this substrate into splayed arm products necessitates unwinding of the terminal heterologous regions. To reveal branch migration activity on its own, a substrate is required that can be processed completely

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Figure 3. FANCM Promotes Branch Migration of Holliday Junctions and Replication Forks (A and B) Schematic representation of the movable Holliday junction (A), the movable replication fork (B), and the heteroduplex products arising from branch migration (BM). Asterisks indicate 32P label at the DNA 50 end. Homologous arms (in black) differ by one base pair. Heterologous arms are drawn with gray and dashed lines. (C) Silver-stained SDS gel showing purified FANCM and K117R FANCM isolated from Sf21 insect cells. (D and E) Autoradiographs showing branch migration of Holliday junctions (D) and replication forks (E). Reactions with FANCM (2 nM) were performed for the indicated periods at 37 C in the presence of ATP (lanes 1–6) or AMP-PNP (lanes 7). Control reactions were carried out in the same way with the ATPase-deficient K117R FANCM protein (2 nM) (lanes 8–14). (F and G) Graphical representation of product formation quantified by PhosphorImaging.

without unwinding activity (Bugreev et al., 2006). To this end, we prepared a synthetic Holliday junction consisting of two homologous and two heterologous arms (Figure 3A). The two opposite homologous arms differed by one base pair to prevent spontaneous branch migration. A replication fork was prepared in a similar way (Figure 3B). Using these substrates, FANCM moved the branchpoint of Holliday junctions and replication forks to dissolution, thereby forming heteroduplex products (Figures 3D and 3E, lanes 1–6). As expected, branch migration activity was strictly dependent on ATP hydrolysis, and it was abolished when ATP was replaced by the nonhydrolysable analog AMP-PNP (Figure 3D, lane 7, and Figure 3E, lane 7). Accordingly, the ATPase mutant protein K117R FANCM (Figure 3C) (Meetei et al., 2005) displayed a greatly diminished branch migration activity on both structures (Figures 3D and 3E, lanes 8–14). Time course analyses showed that the branch migration reactions catalyzed by FANCM proceeded quickly and reached a plateau phase already after 5 min (Figures 3D–3G). In contrast, the reactions catalyzed by K117R FANCM displayed significantly reduced kinetics. Strikingly, 0.2 nM of wild-type FANCM was sufficient to promote efficient branch migration of 0.5 nM of Holliday junctions and replication forks in a 15 min reaction (Figure S2). We noted that, although FANCM appeared remarkably active, the branch migration reactions did not reach completion. We

suspect that this is due to the formation of unproductive protein-DNA complexes, in which the positioning of FANCM relative to the heterologous arms does not permit branch migration. Because FANCM-DNA interactions are highly stable (Figures 2E and 2F), unproductive complexes are likely to stay bound during a 15 min incubation time. FAAP24 Is Dispensable for DNA Binding and Branch Migration Activity of FANCM In vivo, FANCM is part of a multiprotein machine, the FA core complex. FANCM associates directly with FAAP24 via homotypic interactions involving the C-terminal ERCC4 domains of FAAP24 and FANCM (Ciccia et al., 2007). To test whether DNA binding and branch migration activity of FANCM persist in the context of a FANCM/FAAP24 heterodimer, we coexpressed both proteins and isolated them with the same purification scheme as for FANCM (Figure 4A, lanes 1 and 2). To confirm that genuine FANCM/FAAP24 heterodimers were purified, we performed coimmunoprecipitation experiments with an antibody directed against FAAP24. FANCM was coprecipitated with FAAP24 from the FANCM/FAAP24 preparation, but not from the preparation containing only FANCM, suggesting that FANCM and FAAP24 formed a stable heterodimeric complex in the protein preparation (Figure 4A, lanes 3 and 4).

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Figure 4. FAAP24 Is Dispensable for DNA Binding and Branch Migration (A) Silver-stained SDS gel (lane 1) and western blot (lane 2) showing purified FANCM/FAAP24 heterodimers. His-FAAP24 was purified along with Flag-FANCM over anti-Flag agarose and ssDNA-cellulose columns. Immunoprecipitation experiments with an anti-FAAP24 antibody were performed to further confirm the interaction of FANCM and FAAP24 in the preparation (lane 4). A control experiment was carried out with a protein preparation containing FANCM only (lane 3). (B) Silver-stained SDS gel showing purified FAAP24 carrying an N-terminal His tag. (C) DNA binding of FANCM (lanes 2 and 6) was compared with FANCM/FAAP24 heterodimers (lanes 3 and 7) and FAAP24 (lanes 4 and 8). 1.25 nM of proteins was incubated with 0.5 nM of Holliday junctions and replication forks, and the protein-DNA complexes were resolved on 6% polyacrylamide gels. () free DNA; () protein-DNA complexes. (D and E) Autoradiographs showing branch migration of Holliday junctions (D) and replication forks (E). Reactions with 1.25 nM FANCM (lanes 1–5) or 1.25 nM FANCM/FAAP24 (lanes 6–10) were performed for the indicated periods at 37 C in the presence of ATP (lanes 1–4 and 6–9) or AMP-PNP (lanes 5 and 10).

When comparing equimolar amounts of FANCM and FANCM/ FAAP24, neither DNA binding (Figure 4C) nor branch migration activity (Figures 4D and 4E) of FANCM was affected by the presence of FAAP24. Moreover, not even the addition of a 50fold molar excess of purified FAAP24 (Figure 4B) had an influence on branch migration catalyzed by FANCM (data not shown). FANCM Promotes Branch Migration through a 2.6 kb Region of Homology We then wished to challenge the branch migration activity of FANCM with large recombination intermediates (a structures), which contain a Holliday junction (Figure 5A). These intermediates were generated in a RecA-catalyzed strand-exchange reaction between plasmid-based linear duplex and gapped circular DNA (Eggleston et al., 1997). A heterologous block of 1.7 kb, which prevents completion of the strand-exchange reaction, was included for stabilization of the a structure.

First, the association of FANCM with a structures was visualized by electron microscopy (EM). All observed DNA molecules revealed that only one protein particle per DNA molecule was bound and that it was confined to the junction point within the recombination intermediate. No protein-DNA complexes were observed with linear duplex DNA (data not shown). FANCM bound to the DNA substrate at the Holliday junction is shown in Figure 5B. The representative EM image displays a protein particle from which the circular DNA portion and the 2.6 kb homologous and 1.7 kb heterologous arms of the a structure emerge. In a branch migration reaction, FANCM converted almost 60% of the a structures into gapped circular and linear duplex molecules within 2 hr, which implicates translocation of Holliday junctions through a 2.6 kb DNA region of homology (Figure 5C, lanes 1–5). As expected, branch migration was strictly dependent on ATP and did not occur in the presence of AMP-PNP (Figure 5C, lane 6). Time course analyses showed that the branch migration reactions catalyzed by K117R FANCM

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Figure 5. FANCM Dissociates a Structures via Branch Migration (A) Schematic representation of products arising from branch migration. The heterologous block in the a structure is shown in gray. Asterisks indicate 32P label at the DNA 30 end. During branch migration, the Holliday junction translocates along the homologous arm until dissociation of the a structure into gapped circular and linear duplex DNA. (B) Electron microscopy image of FANCM bound to an a structure. A drawing representing the FANCM-DNA complex is depicted next to the EM picture: the short arm (dark gray), the long arm (light gray), and the circular portion (black) of the a structure emerging from FANCM (black) are shown. The scale bar represents 90 nm. (C) Autoradiographs showing dissociation of a structures (0.26 nM) via branch migration. Reactions with FANCM (2 nM) were performed for the indicated periods at 37 C in the presence of ATP (lanes 1–5) or AMP-PNP (lane 6). In lanes 7–12, the same reactions were carried out with the ATPase-deficient K117R FANCM protein (2 nM). (D) Product formation was quantified by PhosphorImaging. It was taken into consideration that the yield of a structures formed in the strand-exchange reaction was only about 70%, and unreacted linear DNA was subtracted as background. Branch migration (BM) was measured by the increase in linear DNA.

displayed significantly reduced kinetics (Figure 5C, lanes 7–12; Figure 5D). We conclude that FANCM is a potent branchpoint translocase. DISCUSSION Whereas the crucial role of the FA/BRCA pathway in the response to replication stress is widely accepted, the molecular function of FA proteins is largely unknown. In this work, we show that purified full-length FANCM binds with high affinity to model Holliday junctions and replication forks. Recently, it has been proposed that FAAP24 may target FANCM and, hence, the FA core complex to DNA replication and repair intermediates (Ciccia et al., 2007). FAAP24 exhibits medium affinity (KD = 3 3 107 M) for branched DNA molecules, such as splayed arm and 30 flap DNA, but does not bind to model replication forks

(Ciccia et al., 2007). Here we show that the affinity of FANCM for branched DNA molecules (KD = 6 3 1011 M–1.2 3 1010 M) is three to four orders of magnitude above the one exhibited by FAAP24. Hence, our results suggest that FANCM itself is endowed with the capacity to target the FA core complex to DNA. Furthermore, we show that FANCM can translocate the branchpoint of model Holliday junctions and replication forks. FANCM has previously been reported to be capable of displacing the third strand of a triple helix (Meetei et al., 2005). Whereas triplex dissociation suggests that FANCM translocates along dsDNA, the branch migration activity of FANCM shown here directly demonstrates that FANCM is a translocase. Taken together, our results show that FANCM is able to specifically recognize and process DNA substrates, which mimic biologically significant replication and repair intermediates. In recent years, several Holliday junction-processing proteins have been identified in mammalian cells. The RecQ helicases

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BLM, WRN, RECQL1, and RECQ5b, and the recombination protein RAD54, are able to branch migrate Holliday junctions in vitro (Bugreev et al., 2006; Constantinou et al., 2000; Garcia et al., 2004; Karow et al., 2000; LeRoy et al., 2005). There are, however, notable biochemical differences between these branch migration proteins. Unlike FANCM, RecQ helicases display little specificity for Holliday junctions and unwind a variety of different DNA structures. Moreover, RecQ helicases can promote branch migration through heterologous DNA, whereas FANCM shows little ability to overcome a thermodynamic barrier caused by short regions of heterology. The phenotypes of cells defective for each of the branch migration proteins BLM, WRN, RAD54, or FANCM differ substantially, suggesting that they are not redundant but act in distinct situations within the cell. Specific protein-protein interactions may determine in which context seemingly identical activities in vitro accomplish different functions in the cell. BLM, together with topoisomerase IIIa, has been implicated in the resolution of double Holliday junctions by a mechanism referred to as double-junction dissolution (Wu and Hickson, 2003). Studies in budding yeast have revealed that Sgs1, the yeast homolog of BLM, together with Top3, the yeast homolog of human topoisomerase IIIa, suppresses crossovers during DNA double-strand break repair (Ira et al., 2003). RAD54, on the other hand, is required for multiple steps during homologous recombination (Heyer et al., 2006). FANCM does not play a critical role in the repair of DNA doublestrand breaks by homologous recombination (Mosedale et al., 2005). Whereas FA cells are exquisitely sensitive to crosslinking agents, accumulating evidence suggests that the FA pathway is more generally required to limit genomic instability caused by diverse lesions during DNA replication (Hinz et al., 2007; Tebbs et al., 2005). One possibility is that FANCM translocates the branchpoint of a stalled replication fork away from the replication block, thereby allowing repair factors to gain access to the lesion. Taken together, one could envision FANCM having three possible roles: (1) a structural function within the FA core complex, which would be required for the monoubiquitination of FANCD2 and FANCI, (2) a structure-specific DNA binding activity, which might target the FA core complex to branched DNA, and (3) a catalytical function, which might allow the remodelling of replication intermediates by branch migration. In this experimental system, we did not observe significant differences in the DNA binding and branch migration activity of FANCM alone and FANCM/FAAP24 heterodimers. Additional studies will be required to understand the biological role of FAAP24. Another important question to be addressed in the future is whether FANCM translocates on DNA in association with, or independently of, the FA core complex. The FA pathway most likely entails multiple molecular transitions, which are far from being understood; our findings might provide a key to further elucidate the FA pathway at a molecular level. EXPERIMENTAL PROCEDURES The cloning of cDNAs and the purification of proteins were carried out as described in the Supplemental Experimental Procedures.

DNA Substrates Sequences and combinations of oligonucleotides used for synthetic DNA structures are depicted in Tables S1 and S2 and described elsewhere (Constantinou et al., 2001). Ten pmol of XO1 was 50 end labeled with [g-32P] ATP and T4 polynucleotide kinase. Cold oligonucleotides were added in 5-fold excess and annealed to XO1. The resulting structures were purified from 6% polyacrylamide gels. For the movable Holliday junction, oligonucleotides XO1 and XM4, and XO2 and XM3, were annealed pairwise first. The two pairs were then incubated together for 30 min at 37 C and for further 30 min at room temperature and purified as described above. For the movable replication fork, XO1 and DS2, and FM and DS1, were annealed separately first and then annealed and purified as described for the movable Holliday junction. The a structures were produced as described (Eggleston et al., 1997). RecA was used to catalyze an exchange of strands between gapped circular pDEA7Z (3000 bp) and PstI-linearized pAKE-7Z (4674 bp) DNA molecules. The linear duplex pAKE-7Z DNA was 30 end labeled with [a-32P] dideoxy ATP and terminal transferase. Before use, the products of the strand-exchange reaction were deproteinized, purified through a Sepharose CL-2B gel filtration column (Sigma) equilibrated with 25 mM Na2HPO4/NaH2PO4 (pH 7.4), 75 mM NaCl and 1 mM MgCl2, and concentrated with a VIVASPIN 2 concentrator (10 kDa cutoff, Vivascience). Biochemical Analysis For EMSA reactions, the indicated amounts of protein were incubated for 30 min at RT with 0.5 nM of 50 -32P-labeled DNA substrates, unless otherwise stated, in a 10 ml reaction containing 25 mM Na2HPO4/NaH2PO4 (pH 7.0), 75 mM NaCl, 5% glycerol, 0.005% NP-40, 0.25 mM EDTA, 1 mM TCEP, and 1 mg/ml BSA. For supershifts, Flag-FANCM was preincubated for 1 hr at RT with 0.8 mg rabbit anti-Flag antibody (Sigma) or 0.8 mg rabbit anti-WHIP antibody (Abcam). DNA binding was analyzed by native PAGE through 6% polyacrylamide gels in TBE. For Scatchard plot analysis, 0.5 nM Flag-FANCM was incubated with different amounts of radiolabeled DNA substrates in the same EMSA reaction conditions as described above. Binding was analyzed by gel electrophoresis and quantified by PhosphorImaging. For branch migration assays with synthetic structures, reactions (10 ml) contained 2 nM protein, except when otherwise stated, 0.5 nM 50 -32P-labeled DNA substrate, 25 mM Na2HPO4/NaH2PO4 (pH 7.0), 75 mM NaCl, 5% glycerol, 0.005% NP-40, 0.25 mM EDTA, 1 mM TCEP, 100 mg/ml BSA, 10 nM XO1 oligonucleotide, 0.5 mM MgCl2, and 1 mM ATP or AMP-PNP, as indicated. Reactions were carried out at 37 C for the indicated periods. Reactions were deproteinized for 20 min at 37 C with 2 mg/ml Proteinase K and 0.4% SDS and resolved by native PAGE through 8% polyacrylamide gels in TBE. For assays with a structures, reactions (10 ml) containing 2 nM protein and 0.26 nM 30 -32P-end-labeled recombination intermediates were carried out in 25 mM Na2HPO4/NaH2PO4 (pH 7.0), 75 mM NaCl, 5% glycerol, 0.005% NP-40, 0.25 mM EDTA, 1 mM TCEP, 100 mg/ml BSA, 10 nM XO1 oligonucleotide, 0.5 mM MgCl2, and 1 mM ATP or AMP-PNP, as indicated. After incubation for the indicated times at 37 C, DNA products were deproteinized and resolved by electrophoresis through 1% agarose in TAE buffer containing 0.5 mg/ml ethidium bromide. Electron Microscopy Five nanomolars of Flag-FANCM was incubated with 0.65 nM a structure in 25 mM Na2HPO4/NaH2PO4 (pH 7.0), 75 mM NaCl, 5% glycerol, 0.005% NP40, 0.25 mM EDTA, and 1 mM TCEP for 15 min at RT. Samples were diluted and washed in 5 mM magnesium acetate and stained with uranyl acetate, as described (Sogo et al., 1987). Protein-DNA complexes were visualized by using a Phillips CM12 electron microscope. Supplemental Data Supplemental Data include Supplemental Experimental Procedures, two figures, and two tables and can be found with this article online at http://www. molecule.org/cgi/content/full/29/1/141/DC1/.

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ACKNOWLEDGMENTS We thank Weidong Wang and Stephen C. West for reagents. This work was supported by the Swiss National Science Foundation. Received: May 2, 2007 Revised: August 22, 2007 Accepted: November 16, 2007 Published: January 17, 2008 REFERENCES

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