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Branch Migration During Homologous Recombination: Assembly of a Ruvab-Holliday Junction Complex in Vitro

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Branch Migration During Homologous Recombination: Assembly of a Ruvab-Holliday Junction Complex in Vitro CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Cell, Vol. 80, 787-793, March 10, 1995, Copyright © 1995 by Cell Press Branch Migration during Homologous Recombination: Assembly of a RuvAB-Holliday Junction Complex In Vitro Kevin Hiom and Stephen C. West tion activity, hexamers of RuvB have been found to associ- Imperial Cancer Research Fund ate with tetramers of RuvA (Mitchell and West, 1994), sug- Clare Hall Laboratories gesting that RuvA and RuvB associate in solution prior to South Mimms, Hertfordshire EN6 3LD DNA binding. England In vitro, the RuvA and RuvB proteins exhibit DNA hell- case activity (Tsaneva et al., 1993; Tsaneva and West, 1994). This observation is of particular interest since it Summary suggests that the RuvB ring structure drives branch migra- tion by a reaction that involves transient strand separation. The RuvA and RuvB proteins of E. coli promote the Other DNA helicases, such as the SV40 large T antigen branch migration or movement of Holliday junctions and E. coil DnaB protein, are also known to form hexa- during genetic recombination and DNA repair. Using meric structures on DNA (Mastrangelo et al., 1989; Reha- small synthetic Holliday junctions in which the cross- Kranz and Hurwitz, 1978), indicating that the structural over point is confined near one end of the DNA mole- arrangement observed with RuvB may be common to a cule, we show that RuvAB-mediated branch migration subset of DNA helicases. occurs with a defined polarity. The assembly of RuvA The biochemical properties of RuvA and RuvB are con- and RuvB on the Holliday junction has been investi- sistent with genetic studies that show that ruv mutants are gated by sedimentation analysis and by DNase I foot- defective in a late step in recombination and the recombi- printing. We find that RuvA protein binds and protects national repair of damaged DNA (Benson et al., 1991). The all four strands of DNA at the crossover point, whereas importance of branch migration as a means to generate RuvB protein binds the DNA asymmetrically. The po- heteroduplex DNA, a process central to the exchange of larity of branch migration is defined by the asymmetric genetic information, led us to investigate the mechanism assembly of the RuvAB branch migration complex rel- of protein-driven branch migration. In this paper we report ative to the junction and is consistent with a model in the detection of a stable RuvAB-Holliday junction com- which RuvAB drives branch migration by passing the plex, which we term the RuvAB prebranch migration com- DNA through the hexameric rings of RuvB. plex. Within this complex, RuvA protein contacts all four strands of DNA at the Holliday junction, whereas RuvB Introduction binds to two arms of the flanking DNA. The structural ar- rangement observed within this prebranch migration com- The DNA damage-inducible RuvA and RuvB proteins of plex indicates that two RuvB ring structures (each ring Escherichia coil are involved in the late stages of genetic encompassing one duplex) interact via RuvA to promote recombination and the recombinational repair of damaged unidirectional branch migration. DNA (Benson et al., 1988, 1991; Lloyd et al., 1984; Sargen- tini and Smith, 1989; Shinagawa et al., 1988). In vitro studies Results have helped define the individual properties and combined functions of these two proteins. The RuvA protein (22 kDa) RuvA Protein Binds the DNA Crossover binds DNA and interacts specifically with Holliday junc- In previous studies, small synthetic Holliday junctions con- tions to form stable RuvA-Holliday junction complexes taining centrally located DNA crossovers were used to (Iwasaki et al., 1992; Parsons et al., 1992). RuvA protein demonstrate branch migration by RuvA and RuvB (Par- is also known to interact with RuvB protein (37 kDa) and sons et al., 1992). Branch migration of these substrates to direct the loading of RuvB onto the Holliday junction occurred in either direction, presumably because the sym- (Parsons and West, 1993). Together RuvA and RuvB pro- metrical nature of the junctions enabled RuvAB to bind in mote the movement of the Holliday junction by a process either orientation. known as branch migration (M011er et al., 1993a; Parsons The specific binding of a DNA crossover by RuvA and et at., 1992; Shiba et al., 1991; Tsaneva et al., 1992b). RuvB can be viewed as the primary step in the assembly Catalysis of branch migration requires ATP hydrolysis, of an active branch migration complex. To investigate the which is provided by the RuvB ATPase activity (Iwasaki structural organization of RuvAB-Holliday junction com- et al., 1989; MOiler et al., 1993b). plexes, we limited the assembly of these complexes to a Electron microscopic studies have shown that RuvB as- single orientation. For this purpose we constructed asym- sembles on duplex DNA as hexameric ring structures in metric junctions by annealing oligonucleotides (88 or 89 which the DNA passes through the hollow core of each ring nt long) to form junctions in which the crossover point was (Stasiak et al., 1994). Though the rings tend to associate in confined near one end of the DNA molecule with flanking pairs on duplex DNA, biochemical studies indicate that duplex arms of 65 bp and 24 bp (Figure 1A). single hexameric rings of RuvB most likely represent the Although RuvA-Holliday junction complexes are stable active form of the protein in the presence of RuvA (Mitchell and can be detected by polyacrylamide gel electrophore- and West, 1994). Under conditions similar to those in sis, previous studies showed that the RuvAB-Holliday which the RuvA and RuvB proteins exhibit branch migra- junction complex could only be detected by this assay Cell 788 A 65nt 24nt I I I -I 5' strand 1 3' strand4 5' X 3' strand 2 3' 5' strand 3 AB ~B lAB AB B iI i~ + RuvA x 3 DNA 6 + RuvB J =< ,I 0 L ", I£ %1=11!i 6 + RuvA + RuvB ii!iiiiiAil i + ATP i'/ili ii ,~ d_ ,====, , , J 5 10 15 20 5 10 15 20 abcd etgh ijkt rnnop FractionNumber Figure 2. DNaselFootprintingofRuvA-HollidayandRuvAB-Holliday Figure 1. Formationof RuvA-Hollidayand RuvAB-HollidayJunction Junction Complexes Complexes In Vitro Four 5'-32P-labeledjunctions (labeledin strand 1,2, 3, or 4) were incu- (A) Diagramof the asymmetric Hollidayjunction. The crossoverpoint bated with RuvA (0.57 I~M), RuvB (4.8 p.M), or both as described in is fixed as shown. ExperimentalProcedures. Reactionmixtures were then treatedwith (B) Glycerol gradient centrifugationof RuvA and RuvAB complexes DNase I, and the products were analyzedby denaturing polyacryl- with the Holiday junction DNA. Binding reactionswere carried out as amidegel electrophoresis.The 5'-32P-labeledstrand is indicated.The described in ExperimentalProcedures using RuvA (0,57 IIM), RuvB position of the DNA crossover for each junction is marked with an (4.8 p.M), or both, as indicated, and the resulting complexeswere asterisk, and the footprints of RuvA and RuvAB are indicated. No analyzed by centrifugationthrough glycerol gradients. The reaction DNase I protectionby either RuvA or RuvB was observedwith linear shown in graph d also contained 1 mM ATP. The junction was 5'-32P duplex DNA. labeledin strand 2. after glutaraldehyde fixation in the presence of the nonhy- Holliday junction complexes were formed prior to addition drolyzable analog of ATP, ATPyS (Parsons and West, of DNase I, we observed a footprint that extended approxi- 1993). Because of the inherent instability of the RuvAB- mately 13 nt on either side of the crossover point. This Holliday junction complexes during polyacrylamide gel pattern of protection was observed on all four strands of electrophoresis, in the following analyses we used a com- DNA (Figure 2, lanes c, g, k, and o; data not shown). Under bination of glycerol gradient centrifugation and DNase I the same experimental conditions, we did not observe any footprinting assays to investigate the binding of RuvA and protection of linear duplex DNA, indicating that the foot- RuvB to the Holiday junction. Incubation of 32P-labeled print was due to structure-specific interactions with the junction DNA with RuvA produced DNA-protein com- Holiday crossover (data not shown). The interaction of plexes that sedimented 1.3 times faster than free junction RuvA with the junction is summarized in Figure 3 and DNA through a glycerol gradient (compare graphs a and shows that RuvA bindsthe Holliday junction symmetrically b in Figure 1 B). Gel analyses of fractions from the glycerol about the crossover point, contacting all four flanking du- gradient confirmed that these complexes were identical plex arms. DNase I footprints of symmetrical Hollidayjunc- to RuvA-Hoilidayjunction complexes observed previously tions, in which the crossover point was located centrally, in band shift assays (data not shown). yielded similar results (data not shown). Using DNase I footprinting, we examined the structure of the RuvA-Holliday junction complex by analyzing the Assembly of a RuvAB-Holliday Junction Complex contacts of RuvA with each of the four DNA strands. Four We next examined the interaction of the RuvAB complex identical junctions were prepared, each uniquely 5'-32P with the Holiday junction. When junction DNA was incu- end-labeled in a different DNA strand. The Holliday junc- bated with both RuvA and RuvB and then analyzed by tions were incubated in the absence or presence of RuvA glycerol gradient centrifugation, we observed a complex and treated with DNase I, and the products were analyzed that sedimented 1.5 times faster than free junction DNA by denaturing gel electrophoresis. In the absence of RuvA, (see Figure 1B, graph c). Unlike the band shift assay re- a region of the DNA corresponding to the position of the ported previously, the stability of this complex required crossover was refractory to DNase I digestion, owing to neither the presence of a nonhydrolyzable analog of ATP steric hindrance of the exchanging DNA helices (Figure nor chemical fixation.
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