RuvA is a Sliding Collar that Protects Holliday Junctions...

doi:10.1016/j.jmb.2005.10.075 J. Mol. Biol. (2006) 355, 473–490

RuvA is a Sliding Collar that Protects Holliday Junctionsfrom Unwinding while Promoting Branch Migration

Daniel L. Kaplan1* and Mike O’Donnell1,2

1Rockefeller University,Laboratory of DNA ReplicationNew York, NY 10021, USA

2Howard Hughes MedicalInstitute, Laboratory of DNAReplication, New York, NY10021, USA

0022-2836/$ - see front matter q 2005 E

Present address: D. L. Kaplan, VaDepartment of Biological Sciences,USA.

Abbreviation used: ssDNA, singlE-mail address of the correspond

[email protected]

The RuvAB proteins catalyze branch migration of Holliday junctionsduring DNA recombination in Escherichia coli. RuvA binds tightly to theHolliday junction, and then recruits two RuvB pumps to power branchmigration. Previous investigations have studied RuvA in conjunction withits cellular partner RuvB. The replication fork helicase DnaB catalyzesbranch migration like RuvB but, unlike RuvB, is not dependent on RuvAfor activity. In this study, we specifically analyze the function of RuvA bystudying RuvA in conjunction with DnaB, a DNA pump that does not workwith RuvA in the cell. Thus, we use DnaB as a tool to dissect RuvA functionfrom RuvB. We find that RuvA does not inhibit DnaB-catalyzed branchmigration of a homologous junction, even at high concentrations of RuvA.Hence, specific protein–protein interaction is not required for RuvAmobilization during branch migration, in contrast to previous proposals.However, low concentrations of RuvA block DnaB unwinding at aHolliday junction. RuvA even blocks DnaB-catalyzed unwinding whentwo DnaB rings are acting in concert on opposite sides of the junction.These findings indicate that RuvA is intrinsically mobile at a Hollidayjunction when the DNA is undergoing branch migration, but RuvA isimmobile at the same junction during DNA unwinding. We presentevidence that suggests that RuvA can slide along a Holliday junctionstructure during DnaB-catalyzed branch migration, but not duringunwinding. Thus, RuvA may act as a sliding collar at Holliday junctions,promoting DNA branch migration activity while blocking other DNAremodeling activities. Finally, we show that RuvA is less mobile at aheterologous junction compared to a homologous junction, as twoopposing DnaB pumps are required to mobilize RuvA over heterologousDNA.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: DNA replication; DNA recombination; Holliday junction; branchmigration; RuvA
*Corresponding author
Introduction

DNA recombination functions in Escherichia colito repair damaged DNA and rescue stalledreplication forks.1 During recombination, a DNAstrand is paired with its homolog from a differentduplex in a reaction catalyzed by RecA working inconcert with other proteins.1 This creates a four-way DNA structure, also called a Holliday junction,

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nderbilt University,Nashville, TN 37232,

e-stranded DNA.ing author:

which is processed by the RuvABC proteins.2 TheRuvA protein initially binds the Holliday junction,and then recruits RuvB protein rings to oppositesides of the junction.3,4 RuvB is a molecular motorthat uses the energy derived from ATP binding andhydrolysis to drive unidirectional movement of thefour-way junction.5 The RuvAB complex thenrecruits RuvC to the junction.6 RuvC is a nucleasethat cleaves the Holliday junction, thereby resolvingit into two duplex DNAs.7

RuvAB has been studied by biochemical andstructural techniques. RuvA binds as a tetramer oroctamer to a Holliday junction. The RuvA proteinhas acidic pins that inhibit binding to double-stranded DNA, thereby targeting the protein toHolliday junctions.8–11 RuvB is a hexameric ring

d.

474 RuvA Promotes Branch Migration

protein with a central channel wide enough toencircle double-stranded DNA.11–13 RuvB alonedoes not bind Holliday junction DNA underphysiological conditions, but after RuvA binds theHolliday junction, RuvA facilitates the assembly oftwo RuvB rings onto opposite sides of the RuvA-junction (Figure 4(a)).14–16 The two RuvB rings arethought to function in concert as DNA pumps thatdrive branch migration of the Holliday junction.15

DnaB functions in DNA replication and is amember of the class of ring-shaped helicases.17–19

DnaB is the primary replicative helicase of E. coli,and unwinds the parental duplex to provide single-stranded DNA (ssDNA) for the replicative poly-merases.20 The DnaB hexamer encircles ssDNAwhile translocating along it, pumping the strandthrough the central channel.21–24 Upon encounte-ring a forked duplex structure, the second DNAstrand cannot fit into the central channel of DnaB,and therefore the continued advance of DnaB alongthe original strand forces its separation from thesecond DNA strand.24,25 It is thought that DnaB,like other hexameric helicases, may act at thereplication fork to unwind parental DNA in thismanner.26–28

The DnaB hexamer can also operate in a mode inwhich it encircles both strands of duplex DNA. Inthis mode, the DnaB does not unwind the DNA.24

However, DnaB actively translocates along theduplex while encircling two DNA strands as itpowers branch migration of Holliday junctions.29

Although this reaction is very efficient in vitro, thein vivo role for DnaB-catalyzed branch migration isunclear. In summary, DnaB unwinds DNA whenencircling one DNA strand, and drives DNA branchmigration while encircling two DNA strands.

DnaB and RuvB have several mechanisticfeatures in common. For example, DnaB and RuvBare both hexameric ring proteins that encircleDNA.2 Furthermore, both DnaB and RuvB utilizeATP binding and hydrolysis to unwind DNA with5 0 to 3 0 polarity.24,30–32 DnaB and RuvB also displaceproteins bound to DNA,29,33 and they both drivebranch migration of Holliday junctions.15,29 Finally,two DnaB rings can bind to opposite sides of aHolliday junction, and work in concert to drivebranch migration of an extended heterologousjunction, like RuvB.34–36

Biochemical studies of RuvA in the past havebeen performed with RuvA in conjunction with itscellular partner, RuvB. The biochemical action ofthe RuvAB complex is thus well studied. However,some intrinsic properties of RuvA during its actionare unclear, as it is most often studied in conjunctionwith RuvB. This leaves unanswered a number ofquestions of how RuvA functions. For example,how does RuvA bind tightly to Holliday junctions,yet become activated to move during branchmigration? Previous proposals suggest that RuvBmust mobilize RuvA bound to a Holliday junction,and that this action is mediated via specific protein–protein interaction between RuvB and RuvA.10

However, it is difficult to study the process that

mobilizes RuvA using only RuvA and RuvB, sincethe activities of these two proteins are dependentupon one another.2 Unlike RuvB, DnaB does notrequire RuvA for activation. Thus, we investigatedhow RuvA moves at a Holliday junction by usingDnaB in conjunction with RuvA. In this study, DnaBis used as a tool to study how RuvA functions, sincethese two proteins do not function together in vivo.

There is an additional advantage to using DnaBwith RuvA to study RuvA function. RuvB ringsbind to opposite arms of a RuvA-bound Hollidayjunction in either of two orientations (Figure 4(a)).Thus, it is difficult to target RuvB loading to aparticular junction arm. In contrast, DnaB loadsonto junction-arm DNA only if a 5 0single-strandextension (5 0 tail) is attached to a particular junctionarm. Thus, unlike RuvB, one DnaB hexamer can beloaded onto a particular junction arm by specificallyadding a 5 0 loading tail.

We find that RuvA binds tightly to Hollidayjunction DNA and blocks DnaB-catalyzed unwin-ding of a Holliday junction. Unwinding activity isblocked even when two DnaB hexamers act inconcert. However, RuvA does not block DnaB-catalyzed branch migration of a homologous Holli-day junction. Hence, RuvA does not need specificprotein activation by RuvB to mobilize in thedirection of branch migration. We present evidencethat suggests that RuvA slides along the Hollidayjunction during DNA branch migration, but notduring DNA unwinding. Interestingly, two DnaBpumps are needed to power migration of RuvAover heterologous DNA, an action that fits nicelywith the physiological architecture of two RuvBpumps straddling the RuvA protein at a Hollidayjunction.

Results

Homologous and heterologous Hollidayjunction DNA substrates used in this study

Holliday junctions in the cell are usually homo-logous, as RecA normally pairs DNA strands of thesame sequence to create the junction. By homolo-gous, we mean that the DNA arms containcomplementary sequences before and after branchmigration. Figure 1(a) shows a homologous Holli-day junction substrate used in this study. Note thatthe substrate is not completely homologous, other-wise the Holliday junction is unstable and canmigrate spontaneously. Thus, the homologoussubstrate used here bears a small degree ofheterology (5 bp) to stabilize the structure andrender it amenable to experimentation. In thereaction shown in Figure 1(a), the Holliday junctionbranch-point migrates a distance of 45 bp, of which40 are complementary, while five are non-comple-mentary.

Heterologous junctions contain DNA sequencethat is non-complementary, and the DNA arm willtherefore become unpaired after branch migration

Figure 1. Homologous and heterologous Holliday junction DNA substrates used in this study. The substrates shownare used in this study to assess RuvA mobility at (a) a homologous and (b) heterologous Holliday junction. (a) Thishomologous substrate is used in Figure 2(a). The 1-2 and 3-4 duplexes are 45 bp in length, and the 1-4 and 2-3 duplexesare 25 bp in length. The 5 0 tail is composed of 30 dT residues. Oligonucleotides used to form this substrate are providedin Table 1. In the reaction shown, the branch-point migrates for 45 bp, of which 40 bp are complementary, and 5 bp arenon-complementary. (b) This heterologous substrate is used in Figure 6(a). The 1-2 and 3-4 duplexes are 25 bp in length,and the 1-4 and 2-3 duplexes are 25 bp in length. The 5 0 tail is composed of 30 dT residues. In the reaction shown, thebranch-point migrates over 19 non-complementary base-pairs.

RuvA Promotes Branch Migration 475

over the heterologous region. Heterologous Holli-day junctions may be found in the cell duringepisodes of DNA damage, when the integrity andsequence fidelity of the DNA are compromised.Thus, the cell may contain heterologous Hollidayjunctions under conditions of DNA damage. In thisstudy, the mobility of RuvA at homologousand heterologous DNA structures are investigated,since either may occur in vivo. Figure 1(b) shows aheterologous junction substrate used inthis study. For this substrate, 19 bp of contiguous,non-complementary DNA sequence are present inthe DNA products after branch migration.

RuvA inhibits DnaB-catalyzed unwinding, butnot DnaB-catalyzed branch migration, of ahomologous junction

Here, we use DnaB as a tool to study how RuvAfunctions at a Holliday junction. In the experimentillustrated by Figure 2(a), DnaB was incubated witha long homologous Holliday junction with a single5 0 loading tail for DnaB (also illustrated inFigure 1(a)). We have shown that with only a 5 0

tail and no 3 0 tail, DnaB tracks on the ssDNA tailand then, instead of unwinding, it travels onto theduplex by encircling both strands and catalyzesbranch migration.35 DnaB was incubated with the

homologous junction for 0.5 min to 4 min at 37 8C.The homologous junction is composed of strands 1,2, 3, and 4. Strand 1 is radiolabeled (asterisk, strand*1). Reactions were quenched and products wereresolved on a native gel, which separates the largerjunction substrate from the smaller branchmigration product that migrates faster. DnaBpromotes substantial branch migration of thissubstrate, as expected (Figure 1(a), accumulationof *1-4 duplex). The model above each gel is used toorient the reader, and was determined from theexperimental gel evidence below. The arrows nextto the gel show the migration distance of DNAproducts as determined by radiolabeled DNAmarkers that were analyzed in the same gel in theexperiments described here. For clarity, however,markers are cropped from the gel images.

In Figure 2(b), the substrate was first incubatedwith RuvA for 1 min, followed by addition of DnaBas in Figure 2(a). Nearly identical results wereobtained in the absence or in the presence of RuvA(compare Figure 2(a) with (b), and view quantifi-cation of product accumulation in Figure 2(c)).Therefore, RuvA does not inhibit DnaB-catalyzedbranch migration of a homologous junction. Thisresult suggests that RuvA is intrinsically mobile ona homologous Holliday junction during DNAbranch migration, and does not require activation

Figure 2. RuvA inhibits DnaB-catalyzed unwinding, but not DnaB-catalyzed branch migration, of a homologousjunction. The model above each gel is used to orient the reader, and was determined from the experimental gel evidencebelow. (a) DnaB acts on a long homologous Holliday junction with one 5 0 tail. The 1-2 and 3-4 duplexes are 45 bp inlength, and the 1-4 and 2-3 duplexes are 25 bp in length. The 5 0 tail is composed of 30 dT residues. Oligonucleotides usedto form this substrate are provided in Table 1. DnaB was incubated with the junction illustrated for the time indicated.Native gel analysis of the reaction is shown. The arrows next to the gel show the migration distance of DNA products asdetermined by radiolabeled DNA markers electrophoresed in the same gel. Markers are cropped from the gel images forclarity. (b) Same as (a), except the substrate is pre-incubated with RuvA for 1 min prior to adding DnaB.(c) Quantification of the accumulation of branch migration product (*1, 4) from (a) and (a). (d) DnaB acts on a longhomologous Holliday junction with one fork. DnaB was incubated with the junction DNA illustrated for the timeindicated. The substrate is identical with (a), except strand 4 contains a 27 bp double-stranded 3 0 tail. Native gel analysisof the reaction is shown. (e) Same as (d), except the substrate is pre-incubated with RuvA for 1 min prior to adding DnaB.(f) Quantification of the accumulation of unwinding products (*2, 3, 4 and *2) from (a) and (b).


through specific protein–protein contacts withRuvB.

The previous experiment demonstrates thatRuvA does not inhibit DnaB-catalyzed branchmigration of a homologous Holliday junction.Does RuvA inhibit other DNA-modulatingreactions at a Holliday junction? To test if RuvAinhibits other DNA remodeling activities, RuvAand DnaB were incubated with a homologousHolliday junction bearing a forked structure atone end (Figure 2(d)). We have shown that DnaBwill unwind a Holliday junction if the loading site is

forked.29 DnaB loads onto the 5 0 tail of this forkedsubstrate. The 3 0 tail of the fork is double-stranded,which promotes DnaB-catalyzed unwinding byaiding exclusion of this 3 0 tail from the centralchannel of DnaB.24 DnaB first unwinds strand 1 ofthis substrate to produce an intermediate productcomposed of strand *2, strand 3, and the 4 duplex(see reaction (i) in Figure 2(d), and the gel band atearly time-points that corresponds to this inter-mediate product). Later in the reaction, DnaBunwinds strand *2 of this intermediate product toyield free strand *2 (see reaction (ii) in Figure 2(d),


and the gel band at later time-points correspondingto free strand *2). (Strand 2 of this substrate islabeled instead of strand 1 to definitively distin-guish unwinding from branch migration. If strand 1were labeled here, DnaB could unwind the 1-4product to yield free strand 1, thereby making itdifficult to distinguish branch migration fromunwinding. The DnaB enzyme is not fully trappedin these reactions, and it may react with the primaryproducts generated to produce secondary reactionproducts.)

This experiment was then repeated, but thesubstrate was first incubated with RuvA(Figure 2(e)). The result shows that RuvA inhibitsthe accumulation of unwinding products substan-tially (compare Figure 2(d) with (e) and see thequantification of unwinding product accumulationin Figure 2(f)). Thus, RuvA inhibits DnaB-catalyzedunwinding of this homologous Holliday junctionsubstrate. This observation suggests that RuvAblocks DNA unwinding at a Holliday junction.

In Figure 3, DnaB was incubated with a Hollidayjunction that is similar to that of Figure 2(d), exceptthe 3 0 tail is single-stranded instead of double-stranded. With a single-stranded 3 0 tail, DnaB nowhas a similar probability of catalyzing unwinding or

branch migration of this substrate, as observedpreviously.29 RuvA blocks DnaB-catalyzed unwin-ding of this substrate, but it does not inhibitDnaB-catalyzed branch migration of this substrate.Thus, RuvA inhibits DnaB unwinding activity, butnot branch migration, at a homologous junction.These results suggest that RuvA is free to move at aHolliday junction during DNA branch migration,while RuvA is immobile at the same Hollidayjunction during DNA unwinding.

We next performed several control experimentsto test if our RuvAB proteins behave as described byother laboratories. RuvA binds to Holliday junc-tions, and then recruits two RuvB hexameric ringsto opposite sides of RuvA. The RuvAB complexthen pumps DNA to drive branch migration. To testif our RuvA and RuvB proteins are able to drivebranch migration, as others have reported, the twoproteins were incubated with a radiolabeled hetero-logous Holliday junction (Figure 4(a)). Branchmigration of this heterologous junction substratewill result in two smaller products (Figure 4(a)).RuvA was incubated with the heterologous junctionfor 1 min at 37 8C, followed by RuvB for 0.5 min to4 min. RuvB rings usually encircle the *1-2 and 3-4duplexes to drive branch migration of this

Figure 3. RuvA inhibits DnaB-catalyzed unwinding, but notDnaB-catalyzed branch migration,of a homologous junction at asingle DNA substrate. The modelabove each gel is used to orient thereader, and was determined fromthe experimental gel evidencebelow. (a) DnaB acts on a longhomologous Holliday junctionwith one fork. The 1-2 and 3-4duplexes are 45 bp in length, andthe 1-4 and 2-3 duplexes are 25 bpin length. Each 5 0 tail is composedof 30 dT, and each 3 0 tail iscomposed of 30 dT. Oligonucleo-tides used to form this substrateare provided in Table 1. DnaB wasincubated with the junction illus-trated for the time indicated.Native gel analysis of the reactionis shown. The arrows next to thegel show the migration distance ofDNA products as determined byradiolabeled DNA markers electro-phoresed in the same gel. Markersare cropped from the gel imagesfor clarity. (b) Same as (a), exceptthe substrate is pre-incubated withRuvA for 1 min prior to addingDnaB.

Figure 4. RuvA does not recruitDnaB to Holliday junctions. Themodel above each gel is used toorient the reader, and was deter-mined from the experimental gelevidence below. (a) RuvAB activityon a heterologous Holliday junc-tion. The numbers represent namesfor each DNA strand. Strand 1 islabeled with 32P at the 5 0 terminus(asterisk). Duplexes 1-2 and 3-4 are45 bp in length, and duplexes 1-4and 2-3 are 25 bp in length. Oligo-nucleotides used to form this sub-strate are provided in Table 1.RuvA was incubated with theheterologous junction illustratedfor 1 min, followed by incubationwith RuvB for the periods of timeindicated. Native gel analysis of thereaction products is shown. Thearrows next to the gel show themigration distance of DNAproducts as determined by radio-labeled DNA markers electrophor-esed in the same gel. Markers arecropped from the gel images forclarity. (b) Same as (a), except RuvAis omitted from the reaction.(c) DnaB acts on the heterologousHolliday junction illustrated in (a).DnaB was incubated with theheterologous junction illustratedfor the time periods indicated.Native gel analysis of the reactionis shown. (d) Same as (c), except thesubstrate is pre-incubated withRuvA for 1 min prior to addingDnaB.


substrate, producing the *1-2 product. This reactionis shown in scheme (i) of Figure 4(a), and confirmedby the dark product band in the gel. Less frequently,RuvB encircles the *1-4 and 2-3 duplexes to drivebranch migration, producing the radiolabeled *1-4duplex product (scheme (ii) of Figure 4(a), and faintbranch migration product in the gel).

Reaction (i) of Figure 4(a) occurs far morefrequently than reaction (ii). The reaction preferencecorrelates with the length of the duplex arms. Thisheterologous junction has arm lengths of 45 bp forduplexes 1-2 and 3-4, and 25 bp for duplexes 1-4and 2-3. The binding site size for RuvAB isapproximately 35 bp on either side of the junction.15

Thus, there is likely competition between these tworeactions, and the longer duplex arms act as betterbinding surfaces for the RuvB rings. In the absenceof RuvA, RuvB alone produces very little product(Figure 4(b)). RuvA alone does not produce anyproduct (not shown), and the substrate does notdisassemble spontaneously in the absence of

protein (not shown). In summary, the RuvABproteins used in this study drive branch migrationof Holliday junctions as expected from the work ofother laboratories.8,15,34,37

Next, we performed an additional control toensure that RuvA does not influence DnaB loadingonto a Holliday junction. We do not expect RuvA toload DnaB, as there is no genetic evidence tosuggest that RuvA recruits DnaB. However, weperformed the following control experiment just tobe sure. DnaB was incubated with the Hollidayjunction lacking a 5 0 tail for 0.5 min to 4 min(Figure 4(c)). Very little product accumulation isobserved (Figure 4(c)), as expected for a substratewith no 5 0 tail. To determine if RuvA recruits DnaBto a Holliday junction, RuvA was incubated withthe junction DNA for 1 min at 37 8C, then DnaB wasadded to the reaction for 0.5 min to 4 min(Figure 4(d)). There is no conversion of substrateto product during this time-frame (Figure 4(d)).Thus, RuvA does not load DnaB at a Holliday


junction, as expected for this control. These resultsallow one to conclude that in Figures 2 and 3, DnaBself-loads on the 5 0 single-stranded region of thesubstrate in a manner independent of the presenceof RuvA.

How does RuvA inhibit DnaB unwinding activityat a Holliday junction in Figures 2 and 3? Oneexplanation is that RuvA binds tightly to thejunction substrate, and creates a physical block toDnaB movement. In support of this idea, it has beendemonstrated that RuvA binds tightly to Hollidayjunctions.16 To test if RuvA binds to junction DNAunder our reaction conditions, an electrophoreticmobility-shift assay (EMSA) was performed. Radio-labeled junction DNA was incubated with RuvAunder conditions that are identical with those usedin our branch migration and unwinding assays(Figure 5(a)). The sample was then analyzed byelectrophoreses in a native polyacrylamide gel toseparate the RuvA–DNA complex from free DNA.As the concentration of RuvA is increased, bandscorresponding to RuvA–DNA complexes increasein intensity (Figure 5(b)). (The appearance of severalRuvA–junction DNA complexes is consistent with

Figure 5. RuvA binds tightly to Holliday junctions.Electrophoretic mobility-shift analysis of RuvA binding aHolliday junction. (a) Scheme for RuvA binding aheterologous junction. The duplex arm lengths are each25 bp. (b) After RuvA incubation for 1 min with theradiolabeled junction illustrated in (a), the reaction isanalyzed by native gel electrophoresis. (c) The data from(b) are quantified and plotted.

published studies, and may correspond to tetra-meric RuvA, octameric RuvA, and higher-orderspecies.16) Quantification of the results of this gel isshown in Figure 5(c). RuvA binds half the junctionDNA at a protein concentration of approximately10 nM. Thus, RuvA binds tightly to Hollidayjunction DNA under our reaction conditions, andthis provides a simple explanation for how RuvAinhibits DnaB unwinding activity.

Tight binding of RuvA to Holliday junctions andthe resulting inhibition of unwinding by DnaB raisean interesting question. Why does RuvA not blockDnaB branch migration activity? One possibleexplanation is that DnaB readily dislodges, ordisassembles, the RuvA multimer from the junctionDNA during branch migration but not unwinding.If this is the case, then DnaB must be far more activein dislodging RuvA when DnaB encircles two DNAstrands during branch migration compared to whenDnaB encircles one strand during unwinding.However, previous studies make this hypothesisunlikely. The rate of DnaB-catalyzed proteindisplacement from DNA is similar for DnaBencircling two DNA strands and for DnaB encir-cling one DNA strand.29 In addition, DnaB is quiteslow to displace tightly bound proteins from DNArelative to the rate of DnaB-catalyzed branchmigration activity.29

An alternative hypothesis to account for theobservation that RuvA blocks DnaB-catalyzedunwinding but not branch migration is that RuvAcan slide along the Holliday junction structureduring branch migration, but not unwinding(Figure 10). For DnaB to catalyze unwinding of anRuvA-bound Holliday junction, RuvA is probablydisplaced, since the four-way junction structure isdestroyed during the reaction (Figure 10(a)). Incontrast, during DnaB-catalyzed branch migrationof an RuvA-homologous junction complex, thejunction structure is preserved continuously (untilthe junction is eventually split when branchmigration reaches the end of the substrate). Thismay enable RuvA to stay bound to DNA through-out the branch migration process (Figure 10(b)).This proposed difference in mechanism, in whichRuvA slides during branch migration but isdisplaced directly off a junction during unwindingmay explain the difference in RuvA blocking ability.In other words, RuvA binds Holliday junctionsstably, and it may be difficult to dislodge thisprotein from an internal position, but during branchmigration RuvA may slide along with the junction.When branch migration is complete and thejunction is split in two, then RuvA wouldpresumably slide off the end of the DNA.

We would like to determine directly whetherRuvA is displaced from DNA or slides along thejunction structure during DnaB-catalyzed branchmigration. However, this cannot be determined byexamining whether RuvA is on or off DNA, asRuvA must fall off the DNA substrate in bothscenarios, whether it is dislodged from the internaljunction structure directly, or whether it slides off


the end only after branch migration is complete. Inboth cases, RuvA is bound to DNA only transiently.Furthermore, whether DnaB dislodges RuvAdirectly, or DnaB slides with RuvA, DnaB andRuvA will be bound to the Holliday junction at thesame time when DnaB loads onto the RuvA-boundHolliday junction. Thus, simply showing that DnaBand RuvA are bound to junction DNA at the sametime will not distinguish between RuvA sliding andRuvA displacement. We next present an indirectmethod to examine whether RuvA is displaced orslides along the junction during DNA branchmigration.

Figure 6. RuvA inhibits branch migration of a heterologouseach gel is used to orient the reader, and was determined fromshort heterologous Holliday junction with one 5 0 tail. Each duOligonucleotides used to form this substrate are provided infor the periods of time indicated. Native gel analysis of themigration distance of DNA products as determined by radiMarkers are cropped from the gel images for clarity. (b) Same1 min prior to adding DnaB. (c) Data from gels shown in (a) aquantified in (c) are all derived from the *1-4 branch migexperiment similar to that shown in (b), except the concentrafixed at 2 min. Open circles show data from a similar experiDnaB incubation times were chosen to roughly match productacting on the junction illustrated in (a). RuvA was incubaincubation with RuvB for the periods of time indicated. (f) Sa

RuvA inhibits branch migration ofa heterologous junction catalyzedby a single DnaB ring

In the experiments of Figure 6 we examine RuvAand DnaB on a heterologous junction substrate tohelp distinguish between the sliding and directdisplacement models for branch migrationpresented above. Studying RuvA action at aheterologous junction also has important biologicalrelevance, since RuvA is likely to encounter regionsof heterology during DNA damage. The junctionsubstrate of Figure 6(a)–(c) is similar to that of

junction catalyzed by a single DnaB ring. The model abovethe experimental gel evidence below. (a) DnaB acts on a

plex is 25 bp in length, and the 5 0 tail is composed of 30 dT.Table 1. DnaB was incubated with the junction illustratedreaction is shown. The arrows next to the gel show the

olabeled DNA markers electrophoresed in the same gel.as (a), except the substrate is pre-incubated with RuvA fornd (b) were quantified and plotted. The products that areration product. (d) Filled triangles show data from antion of RuvA is varied and the time of DnaB incubation isment using the substrate from Figure 2(b) (4 min DnaB).accumulation in the absence of RuvA. (e) RuvA and RuvB

ted with the junction illustrated for 1 min, followed byme as (e), except RuvA is omitted from the reaction.


Figures 2(a)–(c), except the heterologous junction ofFigure 6(a)–(c) will yield unannealed duplex armsonce branch migration is complete, unlike thehomologous junction of Figure 2(a)–(c). The coreregion of the junction has an identical DNAsequence for the substrates used in Figures 2–6.Thus, the DNA-binding site for RuvA is similar inall of these experiments. We have shown elsewherethat with the junction in Figure 6(a)–(c), DnaB loadsonto strand 1 and travels onto the duplex byencircling strands 1 and 4. DnaB then catalyzesbranch migration while encircling strands 1 and 4.29

DnaB encircles two DNA strands and pumps DNAin a similar manner in the junction of Figure 6(a)–(c)and that of Figure 2(a)–(c). Thus, the ability of DnaBto dislodge RuvA from the junction ofFigure 6(a)–(c) should be the same as the ability ofDnaB to dislodge RuvA from the junction ofFigure 2(a)–(c). Hence, if DnaB displaces RuvArapidly during branch migration on a homologousjunction, RuvA should also have little effect onmigration of a heterologous junction, since DnaBshould simply knock it off the DNA and thenproceed on its way.

A different result maybe expected if RuvA slidesduring branch migration. Once branch migrationstarts, the DNA strands in the junction of Figure 6(a)–(c) will become non-complementary, unlike inFigure 2(a)–(c). Thus, RuvA may be expected tohave difficulty sliding over this heterologous junctiononce branch migration starts, because the junctionstructure will become distorted (see Figure 10(c)).RuvA is structurally suited to bind to a four-wayjunction of annealed, duplex DNA.10 If the armsbecome non-complementary, RuvAwill not be able tooptimally accommodate the unannealed duplexarms. Forcing unannealed arms through the RuvAsandwich may be energetically unfavorable. Thestructures of the homologous and heterologousjunctions differ from each other only after branchmigration has begun. Hence, if RuvA behavesdifferently at a heterologous junction compared to ahomologous junction, then RuvA is likely recogniz-ing the change in junction structure that arises onlyonce branch migration starts. In other words, if RuvAblocks DnaB at a heterologous but not a homologousjunction, then RuvA is likely bound to the junctioncontinuously during branch migration (i.e. RuvAslides). Thus, study of the effect of RuvA on DnaBusing the heterologous substrate of Figure 6(a)–(c)may provide indirect evidence of how RuvA actsduring DnaB-catalyzed branch migration of a hom-ologous junction (i.e. is RuvA displaced or does itslide?).

First, we examined the results of DnaB action onthe heterologous junction in the absence of RuvA asa control (Figure 6(a)). DnaB promotes substantialaccumulation of the *1-4 branch migration product,as expected. The *1-4 duplex is a branch migrationproduct, and not the secondary result of strandreannealing. (The half-time of strand 1 annealingwith strand 4 is 70 min, due to an intramolecularhairpin within the region of strand 1 that base-pairs

with strand 4 and slows the rate of strandannealing.29) The additional faint bands inFigure 6(a) arise from DnaB-catalyzed unwindingof the branch migration product, followed by strandreannealing.29

The effect of RuvA on DnaB-catalyzed branchmigration of the heterologous junction is shown inFigure 6(b). The result shows that the branchmigration product is produced far more slowly inthe presence of RuvA (compare Figure 6(a) with (b)).Quantification of all products produced in Figure 6(a)and (b) shows that RuvA substantially inhibits DnaBactivity at this heterologous junction (Figure 6(c)).Thus, RuvA inhibits DnaB-catalyzed branchmigration at the heterologous junction of Figure 6.

The concentration-dependence of RuvA inhibitionof DnaB activity on this heterologous junction wasstudied (Figure 6(d), filled triangles). There issubstantial inhibition of DnaB-catalyzed branchmigration over a wide range of concentrations ofRuvA. This result contrasts markedly with a similarexperiment performed with the homologous junctionsubstrate from Figure 1(a) (Figure 6(d), open circles),where RuvA does not inhibit DnaB over the samerange of concentrations. Thus, RuvA blocks DnaB-catalyzed branch migration of a heterologous junc-tion, but not of a homologous junction. If DnaBdislodges RuvA from the homologous substrate, itshould also dislodge RuvA rapidly from the heter-ologous substrate. However, RuvA inhibits DnaBgreatly on a heterologous junction. Thus, the rapidrate of branch migration of the homologous junctionin the presence of RuvA supports the model wherebyRuvA slides over the homologous Holliday junctionwhen DnaB pushes it (Figure 10(a)).

As an additional control, we tested RuvAB on theheterologous junction, which should result inbranch migration, as the work of other laboratorieshas shown.8,15,34,37 The results demonstrate thatRuvAB is active on this substrate, with roughlyequal accumulation of each branch migrationproduct (Figure 6(e)). Each duplex arm of thissubstrate has a length of 25 bp. The roughly equalaccumulation of each branch migration productcorrelates with the equal size of the duplex arms.Thus, the hypothesis that the length of the arms setsup a competition between the two branch migrationreactions is supported by these data (i.e. results inFigure 4). There is very little activity when RuvA isexcluded from the reaction (Figure 6(f)).

RuvA inhibits unwinding of a Holliday junctioncatalyzed by a single DnaB ring

We next studied the mobility of RuvA at heter-ologous junctions during DNA unwinding. Weincubated DnaB with a heterologous junction bearingboth 50 and 30 tails (a fork) (Figure 7(a)). The resultshows that DnaB unwinds the strand bearing the 50

tail from the rest of the junction (Figure 7(a), scheme(i)). We have shown elsewhere that DnaB drivesbranch migration of this substrate (Figure 7(a),scheme (ii)),29 and this product is observed, as

Figure 7. RuvA inhibits unwin-ding of a Holliday junction cata-lyzed by a single DnaB ring. Themodel above each gel is used toorient the reader, and was deter-mined from the experimental gelevidence below. (a) DnaB acts on aheterologous Holliday junctionwith one fork. Each duplex is25 bp in length, and the 5 0 and 3 0

tails are each composed of 30 dT.Oligonucleotides used to form thissubstrate are provided in Table 1.DnaB was incubated with thejunction illustrated for the periodsof time indicated. Native gel anal-ysis of the reaction is shown. Thearrows next to the gel show themigration distance of DNAproducts as determined by radio-labeled DNA markers electrophor-esed in the same gel. Markers arecropped from the gel images forclarity. (b) Same as (a), except thesubstrate is incubated with RuvAfor 1 min before adding DnaB.(c) Data from gels shown in(a) and (b) were quantified andplotted. (d) Experiments similar tothose shown in (b) were per-formed, except the concentrationof RuvA is varied and the DnaBincubation time is held constant at2 min. Data from native gel ana-lyses were quantified and plotted

as a function of RuvA concentration (open circles). The experiment was then conducted using RuvB in place of DnaB(filled squares).


indicated in Figure 7(a). (The faint band thataccumulates between these products at later time-points corresponds to the *1-2 duplex that resultsfrom unwound strand 1 reannealing with unwoundstrand 2.29)

To determine if RuvA inhibits DnaB-catalyzedunwinding of a heterologous junction, RuvA wasincubated with the heterologous junction beforeadding DnaB (Figure 7(b)). Only faint products areobserved for both unwinding (strand *1) and branchmigration (*1-4 duplex). Therefore, in the presence ofRuvA, both of these products are produced at aslower rate (compare Figure 7(a) with (b)). Quantifi-cation of all products shows that RuvA substantiallyinhibits DnaB activity on this forked heterologousHolliday junction (Figure 7(c)). Thus, RuvA inhibitsboth DnaB-catalyzed unwinding and branchmigration of the heterologous junction, while RuvAactivates RuvB-catalyzed branch migration on thissame substrate (not shown, but similar to the result inFigure 6(e)).

Next, we examined the concentration-depen-dence of RuvA for inhibition of DnaB, and foractivation of RuvB. Experiments were performed asdescribed above, except the concentration of RuvAwas varied and the time was held constant

(Figure 7(d)). Low concentrations of RuvA activateRuvB-catalyzed branch migration (Figure 7(d),filled squares). Half-maximal stimulation of RuvBactivity occurs at approximately 3 nM RuvA. Half-maximal inhibition of single-DnaB ring activityoccurs at a similar concentration of RuvA (between3 nM and 10 nM, Figure 7(d), open circles). Thus, asimilar concentration of RuvA that activates RuvBalso inhibits DnaB. This is the concentration rangein which RuvA binds to the Holliday junction(Figure 5). Thus, binding of RuvA to the Hollidayjunction at low concentrations likely accounts foractivation of RuvB and inhibition of DnaB.

RuvA inhibits double-DnaB ring-catalyzedunwinding, but not branch migration

RuvA inhibits branch migration of a heterologousjunction catalyzed by a single DnaB ring. DoesRuvA inhibit branch migration of a heterologousjunction catalyzed by two opposing DnaB rings?RuvA loads two opposing RuvB rings onto junctionDNA; thus, the effect of RuvA on two opposingDnaB rings may provide further insight into RuvAmechanism during RuvAB function. Recombi-nation processes must sometimes occur over


regions of unmatched sequences, such as duringDNA damage, and it is therefore relevant to test themobility of RuvA over heterologous regions. Othershave demonstrated that RuvAB can catalyze branchmigration over regions of DNA heterology,34,36,38

but the role of RuvA has not been distinguishedfrom that of RuvB in these publications. Below, weonce again use DnaB in conjunction with RuvA tostudy the role of RuvA in heterologous branchmigration.

DnaB catalyzes branch migration of a longheterologous junction if two opposing DnaB ringsare loaded onto the substrate.35 To load twoDnaB rings onto the DNA, a long heterologousjunction was constructed that contains two 5 0 tailson opposite sides of the junction (Figure 8(a)).DnaB rapidly catalyzes branch migration of thissubstrate, as expected, producing the *1,4 duplexproduct (Figure 7(a), scheme (i), and gel). Thereis a secondary reaction, where the *1-4 duplex isunwound by DnaB to yield free strand *1

stant at 2 min. Data from native gel analyses were quantifiedtriangles). A similar experiment was conducted using a loconcentration of RuvA was varied and the DnaB incubation tcatalyzes unwinding of this two-forked substrate (not shownproduct accumulation in the absence of RuvA.

(Figure 8(a), scheme (ii) and gel). Free strand *1increases in intensity for the first 4 min of thereaction as the *1,4 duplex is unwound by DnaB(Figure 8(a), filled squares in graph). The levels of*1,4 duplex are roughly constant in the first 4 minof the reaction (Figure 8(a), open circles ingraph). This result is explained if the rate ofbranch migration, shown in scheme (i), is similarto the rate of secondary unwinding, shown inscheme (ii).

To determine if RuvA blocks this reaction, thesubstrate was incubated with RuvA before addingDnaB (Figure 8(b)). The opposing DnaB ringscatalyze branch migration of this substrate rapidly,even in the presence of RuvA, producing the *1-4duplex product (Figure 8(b), scheme (i) and gel).The concentration of *1-4 duplex continues toincrease during the first 4 min of the reaction(Figure 8(b), open circles in the graph). In fact, thelevels of branch migration product rise higher in thepresence of RuvA than in the absence of RuvA

Figure 8. RuvA inhibits double-DnaB ring-catalyzed unwinding,but not branch migration. Themodel above each gel is used toorient the reader, and was deter-mined from the experimental gelevidence below. (a) DnaB acts on along heterologous Holliday junc-tion with two 5 0 tails. The 1-2 and3-4 duplexes are 45 bp in length,and the 1-4 and 2-3 duplexes are25 bp in length. Each 5 0 tail iscomposed of 30 dT. Oligonucleo-tides used to form this substrate areprovided in Table 1. DnaB wasincubated with the junction illus-trated for the periods of timeindicated. Native gel analysis ofthe reaction is shown. The arrowsnext to the gel show the migrationdistance of DNA products as deter-mined by radiolabeled DNA mar-kers electrophoresed in the samegel. Markers are cropped from thegel images for clarity. The accumu-lation of the *1,4 duplex (opencircles) and free strand *1 (filledsquares) are quantified and plotted.(b) Same as (a), except the substrateis incubated with RuvA for 1 minbefore adding DnaB. (c) Totalproduct accumulation from gelsshown in (a) (open diamonds) and(b) (filled circles) were quantifiedand plotted. (d) Experiments simi-lar to those shown in (b) wereperformed, except the concen-tration of RuvA is varied and theDnaB incubation time is held con-

and plotted as a function of RuvA concentration (filledng heterologous Holliday junction with two forks. Theime is held constant at 30 s (open circles). DnaB primarily). DnaB incubation times were chosen to roughly match


(compare open circles in the graphs in Figure 8(a)and (b)). This may be due to RuvA inhibiting thesecondary reaction of *1-4 duplex unwinding asthe accumulation of free strand *1 is much slower inthe presence of RuvA (see scheme (ii) and comparefilled squares in the graphs in Figure 8(a) and (b)).

To test if RuvA inhibits the unwinding reaction ofscheme (ii) in Figure 8(b), the *1-4 duplex wasincubated with DnaB in the absence and in thepresence of 300 nM RuvA, the same concentrationof RuvA as that used in Figure 8(b) (Figure 9). Thishigh concentration of RuvA slightly inhibits DnaB-catalyzed unwinding of this duplex. Thus, RuvApartially blocks reaction (ii) in Figure 8(b), explain-ing why in the presence of RuvA, free strand *1levels are low and *1-4 duplex product accumulatessubstantially.

The rate of accumulation of all products inFigure 8(a) and (b) is plotted in the graph shownin Figure 8(c). RuvA does not inhibit the rate ofaccumulation of all products. All products inFigure 8(a) and (b) are derived from branchmigration of the junction substrate (reaction (i)).29

Thus, RuvA inhibits DnaB unwinding of the *1-4duplex, but it does not inhibit DnaB branchmigration of the heterologous junction substratewith two 5 0 tails. This observation indicates that twoDnaB pumps overcome RuvA inhibition of branchmigration of a heterologous junction.

Figure 9. RuvA inhibits DnaB-catalyzed unwinding offorked-duplex DNA. (a) DnaB unwinds forked-duplexDNA. The substrate is the primary branch migrationproduct of Figure 8(a) and (b). Oligonucleotides used toform this substrate are provided in Table 1. DnaB wasincubated with the junction illustrated for the timeindicated. Native gel analysis of the reaction is shown.(b) Same as (a), except the substrate is pre-incubated withRuvA for 1 min prior to adding DnaB.

The ability of RuvA at high concentrations tobind to a free forked-duplex structure and slightlyblock DnaB activity, as demonstrated in Figure 9,raises the question of why RuvA does not bind toforked DNA structures when they are part ofjunction DNA in other experiments in this study(Figures 3 and 7), thereby partially inhibiting DnaBactivity. However, the single-stranded tail regionsof strands 1 and 4 in the forked-duplex of Figure 9are not homopolymeric, and thus may formsecondary structures to which RuvA has someaffinity. DnaB can unwind this substrate becausestrand 4 retains enough single-stranded characterfor DnaB-loading. In contrast, the single-strandedregions of the substrates in Figures 3 and 7 arecomposed entirely of dT. These sequences form nosecondary structure, and RuvA cannot bind to them(data not shown). Thus, RuvA inhibition ofunwinding in Figures 3 and 7 is presumed to bethe result of RuvA binding to the DNA branch-point.

RuvA inhibits single-DnaB ring unwinding andbranch migration activity of a heterologous junc-tion, but it does not inhibit double-ring branchmigration activity. We next investigated if RuvAinhibits double-DnaB ring unwinding activity. Weincubated DnaB with a heterologous junction withforks positioned on either side of the junction(Figure 8(d)). DnaB primarily catalyzes unwindingof this substrate (not shown). After 1 min incu-bation with RuvA, DnaB was added for 30 s. Theconcentration of RuvA was varied from 0 nM to300 nM. Quantification of the reaction is shown inFigure 8(d) (open circles), and compared to a similarexperiment using the substrate in Figure 8(b) (filledtriangles, 2 min DnaB). The result shows that RuvAstill inhibits double-DnaB activity on the unwin-ding substrate, but not the branch-migrationsubstrate. We have shown elsewhere that twoDnaB rings work in conjunction at this heterologousjunction to power branch migration. The combinedpower of the two DnaB pumps likely also pushRuvA over non-complementary base-pairs duringbranch migration (Figure 10(d)). However, the needto dislodge RuvA presumably underlies inhibitionof helicase activity, even when two DnaB rings arepresent. Although we cannot rule out the possibilitythat RuvA is dislodged during branch migrationover DNA heterology (Figure 8(d), filled triangles),it is unlikely, given the weak ability of two DnaBpumps to dislodge RuvA during unwinding(Figure 8(d), open circles).

Discussion

RuvA blocks unwinding, but not branchmigration

RuvA binds Holliday junction DNA tightlyin vitro (Figure 5). Once bound to a Hollidayjunction, RuvA inhibits DnaB unwinding activity(Figures 2(f) and 7(c)). Even when two DnaB

Figure 10. Mobilization of the RuvA sliding collar at a Holliday junction by either one or two DNA pumps. (a) RuvAinhibits DNA unwinding at a Holliday junction. If the junction were to be unwound by a helicase, the Holliday junctionwill be destroyed and RuvA will likely be displaced. Thus, tight binding of the RuvA collar to the Holliday junctionblocks unwinding by DnaB when it encircles one DNA strand. (b) RuvA does not inhibit homologous branch migrationcatalyzed by a single duplex DNA pump like DnaB encircling two DNA strands. During branch migration of ahomologous junction, the junction structure is preserved as it translocates. The RuvA collar may slide along with thejunction at little energetic cost. Thus, the RuvA sliding collar is readily mobilized for branch migration at a homologousjunction, and does not require specific protein activation from RuvB. (c) During branch migration of a heterologousjunction, two of the duplex arms will change from annealed duplex to unannealed duplex. The RuvA collar is optimizedfor binding to fully annealed duplex arms; thus, RuvA junction sliding is inhibited by unannealed DNA. (d) Twoopposing DNA pumps mobilize the RuvA sliding collar at a heterologous junction, overcoming resistance of RuvAsliding over unannealed DNA. The RuvA sliding collar works in conjunction with two opposing RuvB rings in vivo.These two RuvB rings may enable the RuvA collar to slide over regions of DNA heterology or DNA lesions.


hexamers act on opposite sides of the RuvA-junction, RuvA continues to block DnaB-catalyzedunwinding activity (Figure 8(d)). Thus, the RuvA-junction is quite stable and resistant to DNAremodeling activity that involves unwinding. Incontrast, RuvA does not block DnaB-catalyzedbranch migration activity on a homologous Holli-day junction (Figure 2(c)). At a heterologousjunction, RuvA blocks single-DnaB ring branchmigration activity (Figure 6(c)), but not doublering activity (Figure 8(d)). Thus, RuvA blocksunwinding activity but not branch migrationactivity. This result is supported further by theexperiment illustrated by Figure 2, in which RuvAblocks DnaB-catalyzed unwinding, but not branchmigration, at the same junction.

At a homologous junction, RuvA blocks DnaBactivity when the helicase surrounds one strand,but RuvA does not block DnaB when the hexamericring surrounds two strands (Figures 2 and 3). Thus,RuvA is highly specific in its inhibition, as RuvA

recognizes whether DnaB is surrounding one DNAstrand or two. RuvA likely evolved this strandspecificity to help ensure that RuvA is mobile onlyin the correct context of RuvB-catalyzed branchmigration. RuvB usually surrounds two strandsin vivo, and RuvA does not block RuvB when RuvBsurrounds two strands to power branch migration.However, if RuvB were to mistakenly surround onestrand, RuvA would probably block RuvB actionjust as it blocks DnaB action. RuvB may mistakenlysurround one DNA strand during extensive DNAdamage with unannealed DNA strands. RuvB hasbeen shown to act as a DNA helicase in vitro whensurrounding one DNA strand,31 and RuvA mayblock this aberrant function at a Holliday junctionin vivo. RuvA may block other helicases at aHolliday junction by this mechanism as well.

The data presented here suggest that RuvA slidesalong homologous junction DNA during branchmigration, but not unwinding. Why does RuvAnot slide along the Holliday junction during


unwinding? In other words, how can RuvAdistinguish whether DnaB is surrounding one ortwo DNA strands? Our previous studies of DnaBsuggest that the ring-shaped protein functions by asimilar mechanism, whether the helicase ringsurrounds one strand during unwinding, or twoDNA strands during branch migration. In eithercase, DnaB primarily pumps one DNA strandthrough the central channel. The primary differencebetween the two modes of action is that, duringunwinding, the strands become separated and onestrand passes outside of the DnaB ring, whereasduring branch migration, both strands pass throughthe DnaB ring.

While it is unclear at present how RuvA candiscriminate between the two modes of DnaBaction, this puzzle may have a basis similar tothat of the replication termination system inbacteria, the Tus–Ter complex. The Tus–Tercomplex blocks DnaB when the helicaseapproaches from one direction (the permissivedirection), but not the other (the non-permissivedirection).39 The Tus–Ter system is remarkablysimilar to the findings here for RuvA, in that,even for helicases that the Tus protein does notinteract with in vivo, Tus blocks helicase actionfrom only one direction.40,41 Although thismechanism is not understood completely, aninteresting model has been proposed by theDixon laboratory.39 In this model, the ssDNAproduced by unwinding as DnaB approachesadheres to the ssDNA pocket in Tus, holding ittight to DNA and preventing DnaB fromdisplacing it. Since Tus is asymmetric, thessDNA pocket faces the duplex in one directionbut not the other. Hence, DnaB approaching fromthe opposite side can displace Tus, since thessDNA produced by the helicase does notinteract with the ssDNA pocket on the otherside of Tus. Perhaps RuvA acts similarly andbinds ssDNA, allowing it to grip and not slide(RuvA is symmetric, and the hypothetical ssDNApocket would be seen by all directions). ThisssDNA is produced only when DnaB acts as ahelicase, allowing RuvA to bind tightly to theDNA side and not slide. But when DnaBencircles duplex DNA, the ssDNA is notproduced and RuvA develops no extra affinityfor DNA and thereby slides as DnaB pushes onit. Ultimately, this speculation may or may not becorrect, but at least it provides a mechanisticbasis for understanding why RuvA slides whileDnaB encircles two strands, and does not slidewhen it acts as a helicase.

RuvA is intrinsically able to slide during branchmigration

As described above, RuvA likely slides duringbranch migration, but not during unwinding. ForDnaB to catalyze unwinding of an RuvA-boundHolliday junction, RuvA must be displaced as thefour-way junction structure is destroyed during the

reaction (Figure 10(a)). In contrast, during DnaB-catalyzed branch migration of an RuvA-homo-logous junction complex, the fully annealed junc-tion structure is continuously preserved, and thusRuvA can stay bound throughout the process(Figure 10(b)). The RuvA collar can simply slidealong all four duplex arms of the junction whileDnaB drives branch migration. Thus, RuvA bindsHolliday junctions stably, and it is difficult todislodge this protein completely from the DNAsubstrate. However, RuvA may simply slide alongthe junction during branch migration. The homo-logous junction used here bears a slight degree ofheterology (five bases); thus, RuvA can tolerate asmall degree of heterology and still slide freely.There is no evidence that DnaB and RuvA functiontogether in vivo. Thus, the observation that RuvA isreadily mobilized by DnaB for branch migrationsuggests that RuvA has an intrinsic capacity formovement on DNA during branch migration.Moreover, our results show that RuvA does notrequire specific contacts with its in vivo proteinpartner, RuvB, to move readily on DNA branchmigration.

It has been proposed that RuvA binds a Hollidayjunction tightly and, upon interaction with RuvB,RuvA is transformed to a weak DNA binder,capable of movement during branch migration.10

Evidence presented here suggests that RuvA bindsto Holliday junctions tightly but yet is freely mobilein the direction of branch migration, without therequirement for activation by RuvB. Why doesRuvA have an intrinsic capacity to slide in thedirection of branch migration? RuvA needs to sliderapidly when functioning with RuvB during branchmigration. Since RuvA has evolved to slide freelyduring branch migration, all of the energy of theRuvB motor can be utilized for branch migration.Thus, RuvA does not impede the function of itsin vivo partner, RuvB. Furthermore, since RuvAremains bound tightly to Holliday junction DNAduring branch migration, RuvA can continue tofunction as a sliding collar to preserve Hollidayjunction structure, and ultimately aid in therecruitment of the RuvC resolvase. Finally, thiswork suggests that RuvA is part of a family ofnucleic acid sliding proteins that grip DNA tightlyyet are also highly mobile (see below).

Comparison of the RuvA collar with other DNAsliding proteins

RuvA is mobilized readily during branchmigration, even though it is not displaced easilyfrom DNA. How can RuvA bind tightly to DNA,but also be readily pushed to slide along it? In thisrespect, RuvA has some similarity to the polymer-ase processivity clamp. In E. coli, the b-slidingclamp confers processivity to the replicative poly-merase.42 During replication, the b-clamp remainsbound to the DNA duplex, sliding along the DNAat a rate of 500–1000 bp/s. However, it is difficult todislodge the b-clamp from the DNA, and an


accessory protein is required to unload theb-clamp.43

The b-clamp forms a ring around the DNA,which enables tight DNA binding while retainingmobility to slide along DNA.44,45 Similar to theb-clamp, RuvA binds DNA topologically byforming a collar or sandwich structure aroundall four arms of the Holliday junction.46 Thus, thetopological binding of RuvA at a Hollidayjunction ensures that RuvA does not dissociatereadily from DNA during branch migration,while still allowing mobility on the DNA armsby sliding. However, there are important differ-ences between the b-clamp and the RuvA collar.While the b-clamp surrounds one DNA duplex,RuvA is a four-way collar that encircles all fourduplex arms simultaneously. Thus, for RuvA, allfour duplexes must slide within it at the sametime (Figure 10(b)). Moreover, the b-dimer ring isa more stable multimer than the RuvA-octamercollar,47 explaining why the b-dimer requires anaccessory protein to crack open the dimer to loadaround DNA, while the RuvA collar canassemble spontaneously around DNA from twotetramers.

The crystal structure of the b-clamp bound toDNA has not been solved, but the structure ofthe b-clamp alone reveals conserved positive andpolar residues within the central channel.44 Theseresidues may make direct or water-mediatedcontact with the DNA duplex, further ensuringthat the protein remains bound to DNA whilesliding for millions of base-pairs during genomereplication. Although RuvA–DNA crystal struc-tures have not been solved at sufficiently highresolution to map protein–DNA interactionsunambiguously, RuvA has conserved positiveand polar residues that may make direct andwater-mediated contact with Holliday junctionDNA.10 Water-mediated hydrogen bonds arelikely to be quite mobile in an aqueous environ-ment. Thus, the nature of chemical contactbetween RuvA or the b-clamp and DNA appearsdesigned for DNA sliding. Moreover, RuvAprimarily contacts the phosphate backbone ofthe DNA, which will maintain a uniformstructure as RuvA slides along the Hollidayjunction. However, the b-clamp may have fewerdirect contacts with DNA than RuvA, explaininghow the b-clamp may slide more freely alongDNA than RuvA. In addition, the RuvA slidingcollar may not slide on its own, but may need apush by a duplex DNA pump for sliding motion.

Other DNA-metabolizing enzymes, such asrestriction enzymes, have been proposed to slidealong DNA.48,49 Thus, protein sliding along DNAin a non-sequence-specific manner may be usedby proteins to accomplish a variety of functionsin the cell. Many nucleic acid-binding proteinsmust traverse along DNA at a very fast rate toaccomplish their function, and DNA sliding is anelegant mechanism for proteins to move quicklyalong DNA without dissociation.

RuvA slides over a heterologous junction withtwo opposing DNA pumps

Recombination processes must sometimes occurover regions of unmatched sequences, such asduring DNA damage, and it is therefore importantto understand the mobility of RuvA over hetero-logous regions. It has been shown that RuvABcatalyzes branch migration over regions of DNAheterology with less efficiency compared to homo-logous DNA,34,36,38 but the role of RuvA in thisprocess was not dissected from RuvB in theseearlier studies. We show in this study that if thejunction is homologous, RuvA is freely mobile toslide over the junction, as discussed above.However, we show also that if the junction isheterologous, the unannealed strands within theRuvA collar inhibit RuvA sliding. DnaB cannotmobilize RuvA at a heterologous junction if theprotein is loaded on one junction arm. However, ifDnaB is loaded onto opposing junction arms, RuvAno longer inhibits DnaB-catalyzed branchmigration. Thus, two opposing DNA pumps canovercome the inhibition to RuvA sliding that DNAheterology creates. RuvA normally functions withopposing RuvB rings. The two opposing RuvB ringsmay enable the RuvA collar to slide over regions ofheterology, consistent with the published mobilityof the RuvAB complex over regions of hetero-logy.34,36,38 Thus, in vivo, two RuvB pumps mayfunction in coordination to mobilize RuvA throughregions of DNA heterology or DNA lesions.

RuvA may function in vivo to stabilize Hollidayjunction structure and limit action to branchmigration

In vivo, a Holliday junction arises afterRecA-mediated DNA recombination. The Holli-day junction structure must be processed tolinear duplexes to restore genomic integrity. TheRuvABC proteins are responsible for this import-ant cellular function, and RuvA is the first ofthese three proteins to bind the Holliday junction.We demonstrate here that RuvA, acting alone,inhibits DnaB unwinding activity substantially bybinding tightly to a Holliday junction.The collision of a replication fork with anRuvA-bound Holliday junction may not be afrequent event. However, the RuvA collar mayfunction in vivo to protect the Holliday junctionfrom the deleterious action of other DNA-metabolizing enzymes until RuvB is recruited tothe Holliday junction. RuvA may thus protect aHolliday junction from helicases, nucleases, andother enzymes that would destroy the Hollidayjunction structure. Furthermore, once RuvB isbound to RuvA and branch migration ensues, theRuvA can work as a sliding collar to allowefficient RuvB-catalyzed branch migration whilepreserving the structure of the Holliday junctionfrom the deleterious effect of other DNA meta-bolizing enzymes.


Materials and Methods

Proteins and DNA

Proteins were expressed in E. coli and purified asdescribed: RuvA, RuvB,50 and DnaB.51 DNA oligonucleo-tides used to construct the substrates in this work aregiven in Table 1, and were synthesized as described.24

Oligonucleotide strands were labeled with 32P at the5 0-end as described.24

Branch migration assays

All manipulations were performed in microfugetubes on ice, and then shifted to 37 8C, unless statedotherwise. Oligonucleotides were annealed to formDNA substrates as described.35 Enzyme reactionswere incubated at 37 8C for the lengths of timeindicated, and contained 1 nM DNA substrate (concen-tration of labeled strand) in 20 mM Tris–HCl (pH 7.5)5 mM ATP, 5 mM creatine phosphate, 20 mg/ml ofcreatine kinase, 10 mM magnesium acetate, 20% (v/v)glycerol, 100 mM EDTA, 40 mg/ml of bovine serumalbumin, 5 mM DTT, in a final volume of 10 ml. Proteinconcentrations were 500 nM DnaB (hexamer), 300 nMRuvA (monomer), and/or 1 mM RuvB (monomer),

Table 1. Sequences of DNA oligonucleotides

Figure Oligonucleot

1(a) 1TE, 2E, 3O,1(b) 1T, 2, 3, a2(a) and (b) 1TE, 2E, 3O,2(d) and (e) 1TE, 2E, 3O, 43(a) and (b) 1TE, 2E, 3O,4(a) and (d) 1E, 2E, 3E,5(b) 1, 2, 3, a6(a) and (b) 1T, 2, 3, a6(e) and (f) 1T, 2, 3, a7(a) and (b) 1T, 2, 3, a8(a) and (b) 1TE, 2E, 3TE8(d) 1TE, 2E, 3TE

1TE, 2TE, 3TE9(a) and (b) 1TE and1 5 0-GAC GCT GCC GAA TTC TGG CTT GCT AGG AC1E 5 0-GAC GCT GCC GAA TTC TGG CTT GCT AGG ACA1T 5 0-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT

TTG ACC CG-3 0

1TE 5 0-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTTTTG ACC CGA TCG CTT AGG TAC GTT AAC C-3 0

2 5 0-CGG GTC AAC GTG GGC AAA GAT GTC CTA GC2E 5 0-GGT TAA CG T ACC TAA GCG ATC GGG TCA ACG2TE 5 0-GGT TAA CG T ACC TAA GCG ATC GGG TCA AC

TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT3 5 0-GAC GTC ATA GAC GAT TAC ATT GCT AGG AC3E 5 0-GAC GTC ATA GAC GAT TAC ATT GCT AGG ACA3O 5 0-GAC GTC ATA GAC GAT TAC ATT GCT AGG ACA3TE 5 0-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT

ACT ATC GCA CGC TTT CGA ACG AGT CTT A-3 0

4 5 0-GCG ATA GTC TCT AGA CAG CAT GTC CTA GC4E 5 0-TAA GAC TCG TTC GAA AGC GTG CGA TAG TCT4O 5 0-GGT TAA CGT ACC TAA GCG ATC GGG TCA ACG4ODS 5 0-GGT TAA CGT ACC TAA GCG ATC GGG TCA AC

TTT TCG ATA TCC ATC CAT CCA TCC ATG CAC-4OT 5 0-GGT TAA CGT ACC TAA GCG ATC GGG TCA AC

TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-4TE 5 0-TAA GAC TCG TTC GAA AGC GTG CGA TAG TC

TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT5DS 5 0-GTG CAT GGA TGG ATG GAT GGA TAT CGA-3 0

unless stated otherwise. RuvA was incubated withsubstrate DNA for 1 min at 37 8C before addition ofRuvB or DnaB, and then incubated further as indicatedin the Figure or described in the Figurelegend. Reactions were quenched upon adding 1 ml ofproteinase K (10 mg/ml), and incubated at 37 8C for anadditional 1 min, followed by addition of 5 ml of 2%(w/v) SDS, 80 mM EDTA. For gel analysis, 5 ml of 15%(v/v) Ficoll (type 400; Pharmacia) and 0.25% (w/v)xylene cyanol FF were added. Samples were snap-frozen in a dry ice/ethanol bath and stored at K20 8C.Before gel electrophoresis, each reaction tube wasincubated at room temperature in a waterbath for4 min to reduce intrastrand base-pairing.

For some experiments, the rate of spontaneous DNAreannealing is fast relative to the time-course of theexperiment. For these reactions, unlabeled oligonucleotideswere added in tenfold excess to inhibit spontaneous DNAreannealing. The DNA traps used in each experiment arelisted in Table 1. Unlabeled DNA trap was addedimmediately before protein addition. High concentrationsof DNA (O500 nM) are required to fully trap DnaB in thesereactions. At these high concentrations, the DNA trapinhibits DnaB activity at the Holliday junction. Thus, we donot fully trap DnaB in this study, but we do use DNA atlower concentrations to trap DNA products generatedduring the reaction to prevent DNA reannealing.

ides used Cold DNA trap

and 4Ond 4and 4O 1TE-4O

ODS, 5DS 2Eand 4OT 2E-3O, and 2Eand 4E 1E-2E, and 1E-4End 4 Nonend 4 1T-4nd 4 1T-4, and 1T-2

nd 4T 1T-4T, and 1T, and 4E 1TE-4E, and 4E 1TE-4E, and 4TE 1TE-4TE4E None

A TCT TTG CCC ACG TTG ACC CG-30

TCT TTG CCC ACG TTG ACC CGA TCG CTT AGG TAC GTT AAC C-30

GAC GCT GCC GAA TTC TGG CTT GCT AGG ACA TCT TTG CCC ACG

GAC GCT GCC GAA TTC TGG CTT GCT AGG ACA TCT TTG CCC ACG

A ATG TAA TCG TCT ATG ACG TC-30

TGG GCA AAG ATG TCC TAG CAA TGT AAT CGT CTA TGA CGT C-30

G TGG GCA AAG ATG TCC TAG CAA TGT AAT CGT CTA TGA CGT CTTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3 0

A TGC TGT CTA GAG ACT ATC GC-30

TGC TGT CTA GAG ACT ATC GCA CGC TTT CGA ACG AGT CTT A-30

TGC TGT CTC ACG TTG ACC CGA TCG CTT AGG TAC GTT AAC C-30

GAC GTC ATA GAC GAT TAC ATT GCT AGG ACA TGC TGT CTA GAG

A AGC CAG AAT TCG GCA GCG TC-30

CTA GAC AGC ATG TCC TAG CAA GCC AGA ATT CGG CAG CGT C-30

TGA GAC AGC ATG TCC TAG CAA GCC AGA ATT CGG CAG CGT C-30

G TGA GAC AGC ATG TCC TAG CAA GCC AGA ATT CGG CAG CGT C30

G TGA GAC AGC ATG TCC TAG CAA GCC AGA ATT CGG CAG CGT C30

T CTA GAC AGC ATG TCC TAG CAA GCC AGA ATT CGG CAG CGT CTTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3 0


DNA products were separated from DNA substrate ina native polyacrylamide gel as described.29 The gels weredried, exposed to a Phosphor-imaging screen, andquantified as described.29 The arrows next to the gelshow the migration distance of DNA products asdetermined by radiolabeled DNA markers in the samegel. Markers are cropped from the gel images for clarity.

Electrophoretic mobility-shift assay

RuvA was incubated with radiolabeled junction DNAfor 1 min at 37 8C under conditions identical with thoseused for branch migration assays. Then 5 ml of 15% Ficoll(type 400; Pharmacia) and 0.25% xylene cyanol FF wasadded to the sample. Protein–DNA complexes wereseparated from free DNA complexes by native gelelectrophoresis. The gel was composed of 4% (w/v)polyacrylamide (29:1 (w/w) acrylamide/bis acrylamide)in a buffer of TBE (90 mM Tris–HCl/borate, 1 mM EDTA,pH 8.0). The running buffer was TBE. The gel wasdeveloped at 150 V at room temperature until the freeDNA was near the bottom of the gel. The gels were dried,exposed to a Phosphor-imaging screen, and quantified asdescribed.29

Acknowledgements

We thank Nicholas Dixon, Taekjip Ha, and NancyHorton for useful discussions regarding this work.We thank Steve West and Ken Marians forproviding expression vectors for the Ruv proteins.Thanks to everyone in the O’Donnell laboratory.This research was supported by grant GM38839from the NIH and by HHMI. D.L.K. was the Leonand Toby Cooperman Fellow of the Damon RunyonCancer Research Foundation (DRG # 1663).

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Edited by R. Ebright

(Received 1 September 2005; received in revised form 25 October 2005; accepted 26 October 2005)Available online 16 November 2005

RuvA is a Sliding Collar that Protects Holliday Junctions...

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