Rad54, a Swi2/Snf2-like Recombinational Repair Protein, Disassembles Rad51:dsDNA Filaments  Jachen A. Solinger, Konstantin Kiianitsa, Wolf-Dietrich Heyer  Molecular Cell  Volume 10, Issue 5, Pages 1175-1188 (November 2002) DOI: 10.1016/S1097-2765(02)00743-8

Figure 1 Topological Assay for Dissociation of Rad51 from dsDNA (A) Schematic representation of Rad51 dissociation assay. Supercoiled dsDNA is relaxed by eukaryotic topoisomerase I (left side). Upon binding by Rad51, dsDNA is unwound and compensatory positive supercoils (+) are introduced. Relaxation of these molecules by topoisomerase I followed by deproteination leads to the formation of highly negatively supercoiled DNA (form X) (upper reaction). Dissociation of Rad51 protein from the DNA by Rad54 protein leads, after treatment with topoisomerase and deproteination, to less negatively supercoiled or even relaxed molecules (lower reaction). (B) Rad54 protein dissociates Rad51 protein from dsDNA in an ATP-dependent manner. Rad51 protein (7.7 μM) was bound to dsDNA (35 μM φX174 RFI) for 30 min at 23°C. Rad54 (0.375 μM) was added, followed after 5 min by the addition of scavenger DNA (63 μM φX174 RFI digested with FspI/SacII). After 5 hr incubation, topoisomerase I (10 units) was added, and dsDNA was relaxed for 1 hr. Reactions were deproteinized and topoisomers were separated on native 0.8% agarose gels with or without 5 μg/ml chloroquine as indicated. X DNA denotes the sum of highly underwound topoisomers, ranging from ΔLk −35 to −60 or less, which are compressed as one band on gels. The concentration of chloroquine was adjusted so that negatively supercoiled DNA (−sc) would retain almost no supercoils and migrate as relaxed species near the nicked circular DNA (nc) (lane 1), while form X DNA would still remain negatively supercoiled (lanes 4, 5, 8, and 10). Under the same conditions, DNA relaxed by topoisomerase I would gain significant amounts of positive supercoils (+sc) due to intercalation by chloroquine. Reactions were performed without ATP (lanes 2 and 3), without scavenger DNA (lanes 4 and 5), without Rad51 (lanes 6 and 7), or containing all components (lanes 8–10). Storage buffer (lanes 1, 2, 4, and 8), Rad54 (lanes 3, 5, 6, and 9), or Rad54-K341R (lanes 7 and 10) was added as indicated. The amount of Rad54 (0.375 μM) and its stoichiometric ratio to Rad51 (1:20) was chosen because it was found to be optimal in stimulating DNA strand exchange (Solinger et al., 2001). The various topological forms are indicated on the left: nicked circle (nc), negatively supercoiled (−sc), positively supercoiled (+sc), and form X DNA (X DNA). Linear dsDNA can be observed near the nicked form because of the incomplete cleavage of scavenger DNA (this band is absent in lanes 4 and 5). (C) Time course of Rad51 removal. Reactions were incubated at 23°C for 0–4 hr as indicated on top. Time courses were carried out with storage buffer (no Rad54) (lanes 1–5) or with Rad54 (lanes 6–10). The different topoisomers are indicated on the left (see B). The lower gel contained 8 μg/ml chloroquine. Molecular Cell 2002 10, 1175-1188DOI: (10.1016/S1097-2765(02)00743-8)

Figure 2 Two-Dimensional Gel Analysis (A) Two-dimensional analysis of topological assay. Reactions were performed exactly as in Figure 1B and separated on native 1.2% agarose gels without chloroquine in the first dimension followed by a second electrophoresis step with 4 μg/ml chloroquine. The positions of supercoiled (sc), nicked (nc), and linear dsDNA are indicated in the first gel showing substrate DNA without topoisomerase I treatment. The nc and linear DNA species appear on all gels. Scavenger DNA is not visible because it is located to the far lower right and was cropped. All reactions, except the first (supercoiled), were incubated with topoisomerase I. The other reactions contained no additional proteins (relaxed) or the proteins indicated on the top of each gel (hRad51 = human Rad51 protein). The position of form X DNA is indicated (X) for the reaction with Rad51 protein. All reactions contained 10 mM ammonium sulfate for direct comparison between reactions with yeast Rad51 and hRad51. (B) Quantification of 2D gels. The regions of the arc that contain X DNA (white bars) (ΔLk < −25), close to naturally supercoiled DNA (dotted bars) (ΔLk ≅ −25), slightly supercoiled DNA (hatched bars) (ΔLk between −25 and −3), and relaxed DNA (black bars) (ΔLk between −3 and +3) were quantified using ImageQuant software. The relative amounts of each species are shown for all reactions from (A): supercoiled (sc), relaxed (rel), Rad54-K341R (KR), Rad54 (54), Rad51 (51), Rad51+Rad54 (51+54), Rad51+Rad54-K341R (51+KR), human Rad51 (h51), human Rad51+Rad54 (h51+54). (C) Fast dissociation of Rad51 filaments by Rad54. A small circular dsDNA molecule was used to visualize Rad51 dissociation in early time points of the reaction. Reactions were performed exactly as in Figure 1B except that pUC19 dsDNA (35 μM) was used as substrate and linearized (PstI) M13mp19 dsDNA (63 μM) as scavenger. Ammonium sulfate was not included. Electrophoresis conditions were as in (A) using 1.8% agarose gels and 60 V for 22 hr in the first dimension followed by 80 V for 22 hr in the second dimension. Controls include supercoiled substrate (without topoisomerase I), relaxed dsDNA (with topoisomerase I but without Rad51 protein), and Rad51 complexes without Rad54 and with Rad54-K341R as indicated (upper row of gels). All controls were incubated for 1 hr. A time course was carried out with Rad51 filaments in the presence of Rad54 protein (lower row of gels). The position of scavenger DNA is indicated (scav.); all other topoisomer species are equivalent to (A). Molecular Cell 2002 10, 1175-1188DOI: (10.1016/S1097-2765(02)00743-8)

Figure 3 Rad51:dsDNA Filaments Are Relatively Stable (A) ATP status at the end of topological assays. Radioactively labeled [32P]-αATP (0.1 μCi) was added to reactions used in Figure 2A. Aliquots of each reaction were analyzed using thin layer chromatography. Control reactions with apyrase were incubated for 30 min (apyr.). Reactions contained no proteins (sc), topoisomerase I only (rel), topoisomerase I and Rad51 (51), or topoisomerase I, Rad51, and Rad54 (51+54). Relative amounts of AMP (hatched bars), ADP (black bars), and ATP (white bars) are shown. Experiments were carried out in triplicate. (B) Addition of ADP does not lead to dissociation of Rad51 filaments from dsDNA. Topological reactions were carried out exactly as in Figure 1C in the absence of Rad54 but without creatine kinase and with the indicated amounts of ADP. The sum of ADP+ATP was the same in all reactions (23 mM). Rad51 filaments were formed in the absence of ADP for 30 min at 23°C. Time points after the addition of ADP are shown: lanes 1–5, no ADP; lanes 6–10, 30% ADP. The bottom gel contained 5 μg/ml chloroquine. Migration positions of topoisomers are indicated on the left (see legend to Figure 1B). (C) Schematic representation of order of addition reactions in the topological assay. The standard reaction is indicated in I. Incubation times are given in minutes. Reaction II, Rad54 was prebound to scavenger DNA; reaction III, Rad51 (1:30 bp) was prebound to scavenger DNA followed by addition of Rad54. (D) Dissociation of Rad51 from dsDNA depends on order of addition. Storage buffer (lanes 1, 3, and 5) or Rad54 (lanes 2, 4, and 6) was added to reactions as indicated. The order of addition (I–III) is shown in (C). The different DNA topoisomers are indicated on the left (for legend see Figure 1B). The lower gel contained 8 μg/ml chloroquine. All reaction conditions were as in Figure 1B except for the order of addition. (E) Rad51 ATPase is stable in high salt. Using the NADH-coupled ATPase assay, rates for Rad51 ATPase activity were determined (100% corresponds to 0.1 min−1). Reactions contained 30 μM dsDNA (φX174) and 2.5 μM Rad51 protein. Rad51 was bound to the dsDNA for 15 min at 30°C before the addition of NaCl. Concentrations of NaCl are indicated in mM. Rates were measured for 1 hr of kinetics. The background (bkg.) for reactions without Rad51 is shown. Error bars indicate the variation during the 1 hr measurement. (F) Rad51 forms stable complexes with dsDNA in high salt. Reactions as in (E) were used to analyze complex formation of Rad51 with dsDNA. Topoisomerase I (5 units) was added to ATPase reactions for 1 hr. Deproteinized reactions were visualized on 1.5% agarose gels with 0.4 μg/ml chloroquine: supercoiled substrate (no topoisomerase I) (lane 1); 0, 50, 75, 100, 125, 150, 200, 250, 300, and 400 mM of NaCl (lanes 2–11); no Rad51 and no salt (lane 12). Molecular Cell 2002 10, 1175-1188DOI: (10.1016/S1097-2765(02)00743-8)

Figure 4 Effects of Rad51 Concentration on Dissociation by Rad54 (A) Titration of yeast Rad51 in topological assay. Reactions were performed as described in Figure 1B with storage buffer (lanes 1–5) and Rad54 (lanes 6–10). The ratios of Rad51 protein per bp of dsDNA are indicated on top. The gel on the right contained 8 μg/ml chloroquine. Different topological forms are indicated (see Figure 1B for description). (B) Titration of Rad51 in ATPase assay. All reactions contained Rad54 (5 nM) and the indicated amounts of Rad51 (protein per bp dsDNA [0.5 μM φX174]). ATPase rates were measured as in Figure 3E. (C) Titration of human Rad51 in topological assay. All conditions were exactly as in (A) to allow direct comparison of the results. Rad51 from yeast (A) was replaced by human Rad51 (C). All reactions in (A) and (C) contained 10 mM ammonium sulfate. Molecular Cell 2002 10, 1175-1188DOI: (10.1016/S1097-2765(02)00743-8)

Figure 5 Rad51 Dissociation from Linear dsDNA (A) Rad54 overcomes restriction enzyme cleavage protection by Rad51 filaments on dsDNA. φX174 dsDNA (17 μM linearized with PstI or SspI) was covered with Rad51 (7.7 μM). Rad54 (0.375 μM) was added and allowed to interact with the complexes for 5 min. After the addition of scavenger DNA (92 μM), reactions were incubated as indicated. The removal of Rad51 by Rad54 was monitored by the cleavage capability of different restriction enzymes (StuI, SacII, and AhdI). A schematic representation of the linear dsDNA (digested either with PstI or SspI) and the location of the restriction enzyme cleavage sites is shown on top. The length of the respective cleavage fragments for φX174 dsDNA linearized with PstI is indicated on the sides of the gel. Time courses of Rad51 removal after the addition of Rad54 are shown for all three enzymes: lanes 1–4, StuI; lanes 5–8, SacII; lanes 9–12, AhdI. (B and C) Quantitation of restriction enzyme cleavage protection assay. Data were obtained using a PhosphoImager. φX174 dsDNA was linearized with PstI (B) or SspI (C). Accumulation of cleaved fragments in a time-dependent manner is shown for StuI (•), AhdI (○), and SacII (*) (black symbols). Controls without Rad54 protein or containing Rad54-K341R were carried out for time points 0 and 6 hr: StuI (•, ▴), AhdI (○, ▵), and SacII (*, +) (gray symbols). Molecular Cell 2002 10, 1175-1188DOI: (10.1016/S1097-2765(02)00743-8)

Figure 6 Rad54 Overcomes Inhibitory Effects of dsDNA Added Early during DNA Strand Exchange Reactions (A) Rad54 enables DNA strand exchange when dsDNA is added early to the reaction. A schematic representation of the DNA strand exchange reaction using φX174 circular ssDNA (ss) and PstI-digested linear dsDNA (ds) is shown on the left. The 5′ ends were labeled with 32P (*). The products of the reaction are joint molecules (jm) and nicked circular DNA (nc). In standard reactions, Rad51 protein (7.7 μM) was incubated with ssDNA (33 μM [nt] φX174) for 15 min. RPA (1.8 μM) was added, and the reaction was kept at 30°C for 30 min. The time course was started by the addition of dsDNA (33 μM [nt] φX174 RFI linearized with PstI) and either storage buffer, Rad54, or Rad54-K341R protein (0.2 μM), respectively. Samples were deproteinized, and reaction products were separated on native 0.8% agarose gels. Standard DNA strand exchange reactions (lanes 1–6) and reactions containing dsDNA from the beginning (ss+dsDNA) (lanes 7–12) were performed. The order of addition is shown on the left. Incubation times are given in minutes. The amounts of Rad51 are indicated: 1:14 nt of ssDNA (lanes 1–3 and 7–9), 1:4 nt of ssDNA (lanes 4–6 and 10–12). Reactions contained storage buffer (control) (lanes 1, 4, 7, and 10), Rad54 (lanes 2, 5, 8, and 11), or Rad54-K341R (lanes 3, 6, 9, and 12). The substrates and products of the DNA strand exchange reaction are indicated on the left side of the ethidium-bromide stained gel. (B) Quantification of reactions in (A). Gels were analyzed on a PhosphoImager. Rad54 (54), Rad54-K341R (KR), or storage buffer (no 54) was added to the reactions. The lower part of the bars (hatched) indicates the amount of nicked circles, and the upper part indicates the amount of joint molecules (the whole bar corresponds to the sum of nc and jm products). (C) Rad54 is unable to stimulate DNA strand exchange reactions carried out by hRad51. The dsDNA was added at the beginning of the reactions as indicated in (A) (ss+dsDNA). Yeast or hRad51 was added in the indicated ratios (protein per nt of ssDNA). Rad54 (54) or storage buffer (no 54) was added to the reactions. The amount of nicked circles is indicated in the lower part of the bars (hatched) as in (B). All reactions were as in (A) but contained 100 mM ammonium sulfate for direct comparison. (D) RPA is critical for stimulation of DNA strand exchange by Rad54 protein. Reactions were carried out exactly as in (A) (ss+dsDNA protocol with a ratio of Rad51 per nt of ssDNA of 1:14). In lanes 3 and 4, RPA was omitted. In lanes 5 and 6, RPA was added first to ssDNA and dsDNA, followed by Rad51 protein after 15 min and Rad54 protein after an additional 5 min. Storage buffer (lanes 1, 3, and 5) or Rad54 protein (lanes 2, 4, and 6) was added to the reactions. The substrates and products of the DNA strand exchange reaction are indicated on the left side of the gel (see [A]). (E) Quantification of reactions as in (D) using a PhosphoImager. Reactions contained Rad54 (54) or storage buffer (no 54). Molecular Cell 2002 10, 1175-1188DOI: (10.1016/S1097-2765(02)00743-8)

Figure 7 Possible Functions of Rad54 Removal Activity during Homologous Recombination Model for the functions of Rad54 protein during homologous recombination. For details see text. Molecular Cell 2002 10, 1175-1188DOI: (10.1016/S1097-2765(02)00743-8)