Volume 29, Issue 1, Pages 141-148 (January 2008) The Fanconi Anemia Protein FANCM Can Promote Branch Migration of Holliday Junctions and Replication Forks  Kerstin Gari, Chantal Décaillet, Alicja Z. Stasiak, Andrzej Stasiak, Angelos Constantinou  Molecular Cell  Volume 29, Issue 1, Pages 141-148 (January 2008) DOI: 10.1016/j.molcel.2007.11.032 Copyright © 2008 Elsevier Inc. Terms and Conditions

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 5′ 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. Molecular Cell 2008 29, 141-148DOI: (10.1016/j.molcel.2007.11.032) Copyright © 2008 Elsevier Inc. Terms and Conditions

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 μM) 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. Molecular Cell 2008 29, 141-148DOI: (10.1016/j.molcel.2007.11.032) Copyright © 2008 Elsevier Inc. Terms and Conditions

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 5′ 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. Molecular Cell 2008 29, 141-148DOI: (10.1016/j.molcel.2007.11.032) Copyright © 2008 Elsevier Inc. Terms and Conditions

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). Molecular Cell 2008 29, 141-148DOI: (10.1016/j.molcel.2007.11.032) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 FANCM Dissociates α Structures via Branch Migration (A) Schematic representation of products arising from branch migration. The heterologous block in the α structure is shown in gray. Asterisks indicate 32P label at the DNA 3′ end. During branch migration, the Holliday junction translocates along the homologous arm until dissociation of the α structure into gapped circular and linear duplex DNA. (B) Electron microscopy image of FANCM bound to an α 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 α structure emerging from FANCM (black) are shown. The scale bar represents 90 nm. (C) Autoradiographs showing dissociation of α 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 α 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. Molecular Cell 2008 29, 141-148DOI: (10.1016/j.molcel.2007.11.032) Copyright © 2008 Elsevier Inc. Terms and Conditions