Plasmid replication initiator interactions with origin13-mers and polymerase subunits contribute tostrand-specific replisome assemblyAleksandra Wawrzycka, Marta Gross, Anna Wasaznik, and Igor Konieczny1

Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822 Gdansk, Poland

Edited by Charles C. Richardson, Harvard Medical School, Boston, MA, and approved June 23, 2015 (received for review March 11, 2015)

Although the molecular basis for replisome activity has beenextensively investigated, it is not clear what the exact mechanismfor de novo assembly of the replication complex at the replicationorigin is, or how the directionality of replication is determined.Here, using the plasmid RK2 replicon, we analyze the protein in-teractions required for Escherichia coli polymerase III (Pol III) ho-loenzyme association at the replication origin. Our investigationsrevealed that in E. coli, replisome formation at the plasmid origininvolves interactions of the RK2 plasmid replication initiation pro-tein (TrfA) with both the polymerase β- and α-subunits. In thepresence of other replication proteins, including DnaA, helicase,primase and the clamp loader, TrfA interaction with the β-clampcontributes to the formation of the β-clamp nucleoprotein complexon origin DNA. By reconstituting in vitro the replication reaction onssDNA templates, we demonstrate that TrfA interaction with theβ-clamp and sequence-specific TrfA interaction with one strand ofthe plasmid origin DNA unwinding element (DUE) contribute tostrand-specific replisome assembly. Wild-type TrfA, but not theTrfA QLSLF mutant (which does not interact with the β-clamp), inthe presence of primase, helicase, Pol III core, clamp loader, andβ-clamp initiates DNA synthesis on ssDNA template containing13-mers of the bottom strand, but not the top strand, of DUE. Re-sults presented in this work uncovered requirements for anchoringpolymerase at the plasmid replication origin and bring insights ofhow the directionality of DNA replication is determined.

DNA replication initiation | polymerase III | β-clamp | Rep | plasmid RK2

DNA synthesis of prokaryotic and eukaryotic replicons re-quires the coordinated action of several enzymes (reviewed

in detail in 1, 2). These enzymes cooperate to form specific nu-cleoprotein complexes during the course of DNA replication.The formation of the initial complex is a result of a replicationinitiation protein (Rep) or origin recognition complex binding todsDNA within the origin of DNA replication initiation (ori). Thisinteraction of the replication initiators with DNA results in ori-gin opening [i.e., destabilization of the DNA unwinding element(DUE)]. Origin opening provides ssDNA for helicase (3, 4),primase (5), and polymerase.It has been demonstrated that during the opening of the

bacterial chromosomal origin (oriC), the chromosomal replica-tion initiator, DnaA, binds to specific sequences (DnaA boxes)(6) and forms a filament on ssDNA (7, 8). Specific interactionbetween DnaA and the DnaB helicase (9, 10) recruits the heli-case and contributes to its loading by a helicase loader, the DnaCprotein (11). Interactions between DnaB and the τ-subunit ofpolymerase (12), as well as DnaB and primase (13), contribute toreplisome assembly at Escherichia coli oriC. The primase re-quires contact with single-stranded DNA-binding protein (SSB)(14) to remain bound to the RNA primer. Disruption of thisinteraction mediated by the polymerase clamp loader leads toprimase displacement (14); β-clamp loading on primed DNA(15, 16); and, finally, interaction of the polymerase core subunitswith the β-clamp–loaded template (14, 17). β-clamp loading is acomplex reaction involving clamp opening and then positioning

around the DNA with the use of the clamp loader (reviewed inref. 18). The scenario for the polymerase assembly at the repli-cation origin is mainly assumed, based on investigations of themechanism of leading and lagging DNA strand synthesis, con-ducted with in vitro assays on primed circular DNA but not onsupercoiled templates. It is not clear how the replisome is as-sembled on supercoiled dsDNA after origin opening andwhether the helicase interactions with primase and τ-subunit arethe only factors contributing to de novo replisome assembly atthe replication origin.In case of bacterial plasmids, involvement of both the plasmid-

encoded Rep and the host-encoded replication initiator DnaAwas reported as essential for origin opening and helicase com-plex recruitment (19–21). DNA replication of the broad-host-range plasmid RK2 (reviewed in ref. 19) is initiated by the RK2plasmid encoded Rep protein (TrfA), which binds to direct re-peats (iterons) localized at the plasmid’s replication origin (oriV)(22) (Fig. 1A). In contrast to DnaA (23), TrfA, as well as otherplasmid Reps, does not contain a DNA binding domain (DBD).Instead, the plasmid Reps are similar to eukaryotic replicationinitiators and contain a winged helix (WH) domain for DNAinteraction (24, reviewed in 25). TrfA interaction with the iteronsleads to origin opening assisted by host HU and DnaA proteins(26). TrfA plays a crucial role in DnaB helicase recruitment andpositioning at the AT-rich region of the oriV (27, 28). In contrastto DnaA protein, no data for plasmid Rep filament formation onssDNA have been provided to date. Recently, it was shown thatTrfA interacts with ssDNA containing 13-mer sequences of one

Significance

Research on DNA replication initiation has not revealed the ex-act mechanism for replication complex de novo assembly at theorigin or how the directionality of replication is determined. Todate, no evidence for direct involvement of a replication initia-tion protein (Rep) in the process of polymerase recruitment hasbeen reported. This work demonstrates that a plasmid Rep, inaddition to its already described functions in origin opening andhelicase recruitment, can serve as a DNA polymerase anchoringfactor. Through its interaction with 13-mer sequences on onestrand of initially unwound DNA and interactions with thesubunits of DNA polymerase, the initiation protein facilitatesstrand-specific replisome assembly at the replication origin. Thisstep determines the direction of DNA replication.

Author contributions: I.K. designed research; A. Wawrzycka, M.G., and A. Wasaznik per-formed research; A. Wawrzycka, M.G., and I.K. analyzed data; and A. Wawrzycka and I.K.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. Email: igor.konieczny@biotech.ug.edu.pl.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1504926112/-/DCSupplemental.

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strand of the plasmid origin DUE (29). Interestingly, the specificmotif (QL[S/D]LF) determining interaction with the β-clamp hasbeen identified in plasmid Reps (30); however, the relevance ofthe interaction between the β-clamp and Reps has not beenestablished. The QAMSLF motif (related to QLSLF) wasidentified in the δ-subunit of the clamp loader, and it has beenshown to be required for δ-interaction with the β-clamp andclamp loading (16). In this work, we investigate the significanceof the β-clamp interaction with the TrfA replication initiator inthe process of RK2 plasmid DNA replication initiation. We alsoexamine the requirements for the assembly of the replicationcomplex at the plasmid origin.

ResultsTo address the question concerning the significance of TrfAinteraction with the β-clamp, we constructed TrfA variants withmutations in the QLSLF motif that were expected to disrupt thisinteraction. With the use of an ELISA test, it was previouslyshown that LF deletion within the QLSLF motif results in TrfAdefective in β-clamp binding (30). Using PCR-based site-directedmutagenesis, we constructed plasmids carrying genes for TrfAΔLF and TrfA F138A (where Phe in the QLSLF motif was

changed into Ala). Wild-type TrfA (wt TrfA) and TrfA variantswith alterations within the QLSLF motif were purified by affinitychromatography (Materials and Methods). To assess the qualityof the purified proteins, we performed an analysis of the iso-thermal CD spectra (Fig. S1) and calculated the content of therespective secondary structures for each TrfA variant. The re-sults did not reveal any substantial differences in the secondarystructure’s content of the analyzed TrfA variants in comparisonto the wt TrfA. We then determined how the TrfA variantsinteracted with the β-clamp using surface plasmon resonance(SPR) (SI Materials and Methods and Fig. S2). The wt TrfAimmobilized on a CM5 sensor chip interacted with the β-clamp,whereas TrfA ΔLF–β-clamp complex formation was severelyimpaired. Under the same experimental conditions, TrfA F138Ainteracted with the β-clamp, although slightly less efficiently thanwas observed for the wt TrfA.To determine whether the mutations in the QLSLF motif that

altered TrfA’s interaction with the β-clamp influenced RK2DNA synthesis, we tested the replication activity of the purifiedTrfA variants in an in vitro replication assay. The test was basedon E. coli cell crude extract (FII) that allows replication ofsupercoiled dsDNA in the presence of the plasmid replicationinitiator, TrfA. The soluble FII extract contains all proteinsnecessary for plasmid DNA synthesis, including polymerase III(Pol III) holoenzyme and chaperones for TrfA activation. DNAsynthesis in this assay, measured as the total amount of in-corporated nucleotides, reached a maximum when 90 nM wtTrfA was added to the assay mix and was inhibited by largeramounts of this protein (Fig. 1B). Based on the amount of nu-cleotides incorporated into the template, we calculated that 25%of DNA templates were typically copied, similar to results pre-sented by others during experiments with an oriC in vitro system(31, 32). TrfA ΔLF was defective in DNA synthesis; replicationreactions carried out in the presence of varying amounts of thismutant protein remained at background levels. Replication ac-tivity of the TrfA F138A mutant was reduced in comparison towt TrfA but showed a similar activity profile with a peak at90 nM protein and an inhibition of DNA synthesis at largeramounts of protein. Similar results (Fig. 1C) were obtained whenwe performed in vitro replication assays using purified enzymesand a supercoiled dsDNA oriV template according to the methodof Konieczny and Helinski (28), with modifications (SI Materialsand Methods).The in vitro replication results were consistent with the rep-

lication results obtained in transformation efficiency tests. Plas-mid pSV16 was used to transform E. coli cells containing pATplasmid encoding one of the trfA gene variants: wt trfA, trfAΔLF,or trfAF138A. Plasmid pSV16 contains the RK2 origin of repli-cation but not the gene for TrfA; thus, transformant colonygrowth indicates that the pAT encodes a replicatively active formof TrfA. When E. coli carrying pATwt trfA was transformed withpSV16 plasmid, colonies were obtained with a transformationfrequency of 3.8 × 104 (Fig. 1D). No colonies were observedwhen E. coli cells containing plasmid pATtrfAΔLF were trans-formed with pSV16. When E. coli containing pATtrfAF138A wastransformed with pSV16, the obtained transformation frequencywas slightly lower compared with the transformation frequencyobserved with pATwt trfA encoding wt TrfA. In a control ex-periment, when pBBR1MCS2 plasmid (Table S1), replicatingindependent of the TrfA, was used instead of pSV16, we ob-served colony growth of E. coli strains with all three pAT variantstested (Fig. 1D).Our experiments revealed that besides the defects in TrfA

interaction with β-clamp, the analyzed mutations within theQLSLF motif result in a deficiency of plasmid DNA synthesisboth in vitro and in vivo. We then asked at which stage of rep-lication initiation the ΔLF and F138A mutations caused theobserved deficiencies of the in vitro and in vivo replication.

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Fig. 1. TrfA ΔLF is not active in RK2 DNA replication either in vitro or invivo. (A) Plasmid RK2 minimal origin of replication. The scheme presents theRK2 origin (oriV) region comprising the cluster of 17-bp direct repeats(iterons), four DnaA boxes, and the DUE region with four 13-mers. (B) Invitro replication with a crude extract (FII) prepared from E. coli C600. (C) Invitro DNA replication reaction reconstituted with purified proteins (Recon.system). (B and C) Both in vitro replication experiments were establishedwith increasing amounts of wt TrfA or TrfA mutants as noted (0, 30, 60, 90,120, 150, 210, and 300 nM) (results are presented from n = 3 replicates, withthe SD shown). (D) TrfA in vivo activity test. Plasmid pSV16 containing oriVwas used to transform E. coli DH5α cells with plasmids carrying variantsof trfA as indicated. The transformation frequency is reported as thenumber of colony-forming units per 1 μg of supercoiled plasmid pSV16DNA averaged from three independent experiments, with the SD shown.

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To answer this question, we examined the individual steps ofthe initiation process in vitro. First, we tested if mutations withinthe QLSLF motif might affect TrfA interaction with DNA. TrfAbinds to oriV iterons as a monomer (33), and chaperone proteinsare required for TrfA activation by converting TrfA dimers tomonomers (34, 35). Thus, before the DNA interaction tests, weused the E. coli ClpX chaperone to activate the purified TrfAvariants (Materials and Methods). After incubation with thechaperone, we used a gel mobility shift assay (GMSA) (SI Ma-terials and Methods) to analyze binding of the ClpX-activatedTrfA variants to fluorescently labeled dsDNA fragments con-taining a five-iteron sequence of RK2 oriV. Our experimentsrevealed no substantial differences among the tested proteins intheir ability to form nucleoprotein complexes; TrfA ΔLF andTrfA F138A bind to the DNA fragment and produce retardedbands as efficiently as wt TrfA (Fig. S3 A–C). It must be pointedout that, as has been previously demonstrated, the ClpX does notinteract with DNA (36), which we also verified during our ex-periments (Fig. S3D).Because TrfA is involved in DnaB helicase complex formation

at oriV (27, 28), we decided to test if the TrfA ΔLF and TrfAF138A, which were defective in replication, were able to recruitthe helicase. Using gel filtration on a Sepharose CL-4B column(GE Healthcare), we tested helicase complex formation on asupercoiled DNA template containing oriV (pKD19L1) in thepresence of ClpX-activated TrfA variants (Fig. 2A). In additionto supercoiled DNA, TrfA variants, and DnaB, the reactionmixture contained DnaA and DnaC proteins, which are requiredfor helicase complex formation at oriV (28). Under these ex-perimental conditions, plasmid DNA was found in the column’svoid volume fractions (Fig. 2A, Top). Other panels of Fig. 2illustrate DnaB detected by a protein-specific antiserum inconsecutive fractions collected from the column. In a controlexperiment, when wt TrfA was not added to the reaction mix-ture, we did not detect DnaB in the void volume, which indicateda lack of helicase at oriV in the absence of TrfA. In contrast,DnaB protein was observed in void volume fractions when thereaction mixture contained any of the tested variants of TrfA,showing that the TrfA ΔLF supports helicase complex formationon RK2 DNA as well as the wt TrfA (Fig. 2A). TrfA F138Asupports helicase complex formation less efficiently. In controlexperiments, we did not detect DnaB in void volume whenpUC18 supercoiled DNA or no DNA was present in the reactionmixture (Fig. 2A).It has been demonstrated that in addition to DnaB loading,

TrfA is involved in helicase activation at oriV (28). Thus, for-mation of the DnaB helicase complex on DNA might not nec-essarily result in helicase activity. We therefore tested the TrfAvariants as helicase-activating factors. E. coli DnaB helicase ac-tivity on supercoiled RK2 plasmid DNA template was identifiedelectrophoretically by the formation of an extensively unwoundform of the supercoiled plasmid DNA, designated FI* (28). Theformation of the FI* form is a result of the combined activities ofplasmid and bacterial Reps, with the presence of an active formof TrfA being essential. ClpX activation of TrfA variants wasperformed as for the previous in vitro tests, and the other re-action components were then added. Experiments were carriedout with increasing concentrations of the TrfA variants (Fig. 2B,lanes 2–6; lane 1 is the reaction mixture without added TrfA). Inagreement with previous results (34), in the presence of ClpX-activated wt TrfA, we observed the FI* DNA band that indicateshelicase unwinding activity. The FI* form of DNA was also ob-served in the presence of both TrfA mutants tested, TrfA ΔLFand TrfA F138A; however, TrfA F138A was slightly less efficientin helicase activation (Fig. 2B).To acquire helicase unwinding activity on plasmid DNA, a

number of steps, including the formation of an initial complex,origin opening, and helicase complex assembly and its activation,

have to be accomplished in the presence of an active form of TrfA.Our results indicate that TrfA ΔLF, as well as TrfA F138A, pro-motes helicase activation but not plasmid DNA synthesis. Theseresults, together with the confirmed TrfA–β-clamp interaction,strongly suggest a role for TrfA during the assembly of the repli-some complex at the plasmid replication origin. To further ex-amine TrfA’s role in replisome assembly, we studied the formationof nucleoprotein complexes on supercoiled RK2 plasmid DNAusing gel filtration with a fluorescently labeled β-clamp (Fig. 3). Asin previous experiments, wt TrfA, TrfA F138A, or TrfA ΔLF wasinitially activated by incubation with ClpX. Reaction mixtures wereprepared as for in vitro replication assays using purified enzymes(SI Materials and Methods). After incubation, the reaction mixtureswere applied on a Sepharose CL-4B column and the collectedfractions were analyzed for the presence of DNA and fluo-rescently labeled β-clamp. A fluorescent signal in the void vol-ume fractions containing plasmid DNA would indicate that theβ-clamp was found in a nucleoprotein complex. In the presenceof wt TrfA, the fluorescent signal was detected in the void vol-ume fractions (Fig. 3A), whereas when the TrfA ΔLF variant wasused, fluorescence remained at background levels in the voidvolume. The observed fluorescence was significantly reducedwhen TrfA F138A was tested under the same conditions (Fig.3A). We estimated that when wt TrfA was present in the re-actions, ∼15% of the total amount of the β-clamp used for theexperiment was detected in DNA-containing fractions. It should

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Fig. 2. In the presence of TrfA ΔLF or TrfA F138A, the DnaB helicase com-plex is loaded and activated on RK2 DNA. (A) Formation of nucleoproteincomplexes was studied using gel filtration as described in SI Materials andMethods. Reaction mixtures containing each of the ClpX-activated TrfAvariants (120 nM) if present, DnaB (200 nM), DnaA (7.5 nM), DnaC (1.1 μM),and pKD19L1 DNA (2.3 nM) were used. A 20-min incubation was followed bygel filtration. Fractions (80 μL) collected from the column were analyzed bySDS/PAGE and Western blotting for the presence of DnaB and by agaroseelectrophoresis for the presence of DNA. (Top) Plasmid DNA was found invoid volume fractions. (B) E. coli DnaB helicase activity on RK2 DNA wasdetermined by the FI* assay. The position of the FI* DNA band, indicatinghelicase activity, is marked with arrows. Reaction mixtures were as describedin SI Materials and Methods with increasing TrfA concentrations (0, 30, 60,90, 120, and 150 nM in lanes 1–6, respectively).

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be pointed out that a similar efficiency was obtained with theDnaA-dependent reaction on oriC-containing templates (Fig.S4). We also performed control experiments where reactionmixtures contained only fluorescently labeled β-clamp and RK2plasmid DNA or all components except TrfA. In both cases, thefluorescence signal did not appear in void fractions (Fig. S5 E

and F). These results indicated that TrfA, with an intact QLSLFmotif, is specifically required for β-clamp complex formation atthe RK2 plasmid origin. We then determined what other pro-teins in the reaction mixture were indispensable for the β-clampcomplex formation with supercoiled RK2 plasmid DNA. Afluorescent signal was not detected in void fractions in the ab-sence of all of the other Pol III subunits (Fig. 3B), γδδ′χψ (Fig.3D), DnaA (Fig. 3E), DnaB (Fig. 3F), or primase (Fig. 3G). Thepresence of Pol III core subunits (Fig. 3C) or gyrase (Fig. 3H),however, was not essential for β-clamp nucleoprotein complexformation on RK2 plasmid DNA, because we still detectedfluorescent signal in the void volume in the absence of either ofthese factors. When GTP, UTP, and CTP and/or dNTPs wereomitted from the reaction mixture, we still observed a fluores-cent signal in the void fractions (Fig. S5 B–D). Because β-clampnucleoprotein complex formation was dependent on TrfA andγδδ′χψ in our gel filtration experiments, we tested how thecomposition of clamp loader affects the RK2 DNA replication.In vitro replication assays in a reconstituted system with variouslyassembled clamp loaders were performed (Fig. S6A). The re-actions were performed with wt TrfA and TrfA ΔLF. A sub-stantial reduction of the replication activity was observed in theabsence of τ-subunit, although not much difference in replicationactivity was observed when the reaction was conducted as in Fig.1C, containing both τ and γ, in comparison to the reaction withclamp loader lacking γ (Fig. S6A). With TrfA ΔLF or withoutTrfA at all, we detected only a background level of DNA syn-thesis. To test if TrfA can affect ATPase hydrolysis by a clamploader, we performed a clamp loader ATPase activity assay inthe presence of primed DNA (Fig. S6B). No influence of wt TrfAor TrfA ΔLF was observed.To analyze the mechanism of replisome assembly at the RK2

plasmid origin further, we coupled the gel filtration experimentwith immunodetection of the α-subunit of the Pol III core (Fig.4). After incubation, reaction mixtures consisting of bacterialcrude extracts (FII), RK2 supercoiled DNA, and TrfA variantswere applied on a Sepharose CL-4B column and fractions wereimmunoanalyzed. The α was detected in void volume fractionswhen wt TrfA (Fig. 4A) or, surprisingly, the TrfA F138A orTrfA ΔLF mutant (Fig. 4 B and C) was present in reactionmixtures. In control experiments, when TrfA or DNA wasomitted or pUC18 was used instead of RK2, α was not detectedimmunologically in the void volume (Fig. 4 D–F). When wetested for β-clamp nucleoprotein complex formation under thesame experimental conditions, the protein was detected in thepresence of wt TrfA and oriV DNA (Fig. 4G). When TrfA wasomitted or TrfA ΔLF was present in the incubation mixture, weobserved almost no absorbance signal derived from immuno-logical assay. The observed absorbance signal was significantlyreduced when TrfA F138A was used (Fig. 4 G–I). The β-clamptracking in crude extract was in accordance with experimentsperformed for reconstituted reactions (Fig. 3A). Also, when we useda reconstituted system for α-tracking, consistent with the resultsobtained in crude extract, α was eluted in void fractions in thepresence of either wt TrfA or TrfA ΔLF (Fig. S4 D and E). Re-gardless of the system used, the α was found in the nucleoproteincomplex formed on RK2 DNA in the presence of the TrfA variantsdefective in interaction with the β-clamp (Fig. S2) and not pro-moting the β-clamp complex formation at the plasmid origin. Thisunexpected finding raised a question concerning the mechanism ofthis interaction.To address the issue of how α interacts at oriV without the

β-clamp being loaded onto the plasmid DNA, we performed anELISA with α-specific or β-specific antisera (SI Materials andMethods and Fig. S1 C and D). Interestingly, when wt TrfA wasimmobilized on plates and increased concentrations of α wereapplied, we clearly detected an interaction (Fig. 5A). Furthermore,TrfA ΔLF and TrfA F138A interacted with α as efficiently as wt

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Fig. 3. TrfA is required for β-clamp nucleoprotein complex formation on RK2plasmid DNA. Nucleoprotein complex formation in the reconstituted system wasstudied using a gel filtration-based assay. (A) Reaction mixtures containing wtTrfA (120 nM), TrfA F138A (120 nM), or TrfA ΔLF (120 nM) were activated withClpX (870 nM), and DnaA (450 nM), DnaB (200 nM), DnaC (1.1 μM), gyrase(13 nM), HU (110 nM), SSB (150 nM), primase (60 nM), and pKD19L1 (2.3 nM)template DNA were then added. (B–H) Reaction mixtures were prepared asdescribed for A, except that indicated components were omitted. A 20-min in-cubation was carried out to form a helicase complex, and β-clamp labeled withAlexa 488 fluorescent dye (17 nM) and E. coli Pol III core τ-subunits [αeθτ (19 nM)and γδδ′χψ (2.5 nM)] were then added. After 15 s, the reaction was quenched byadding 5 units of hexokinase to remove ATP (51), followed by gel filtration on aSepharose CL-4B column. The collected fractions (40 μL) were analyzed for thepresence of the Alexa 488 β-clamp by measuring fluorescence and for thepresence of DNA by agarose electrophoresis. (Top) Plasmid DNA was found invoid volume fractions only (as labeled).

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TrfA (Fig. 5A). Consistent with our SPR analysis (Fig. S2), weobserved that wt TrfA interacts with the β-clamp, whereas TrfAΔLF is deficient in β-clamp binding (Fig. 5B) and TrfA F138Ainteraction with the β-clamp was reduced by half compared withwt TrfA (Fig. 5B). We were also able to detect an α–β-clamp in-teraction (Fig. 5C).These experiments revealed a network of interactions between

TrfA, β-clamp, and α (Fig. 5D). Interestingly, a β-clamp bindingmotif related to the one found in TrfA is also present in α. Todetermine if the identified interactions were competitive, weperformed two types of ELISA tests. In the first one, wt TrfAwas immobilized on the plate and incubated with mixturescomposed of β-clamp and α in different molar ratios (Fig. 5E).By using β-specific antiserum, we detected a TrfA–β-clampcomplex even when there was an excess of α. For the secondELISA test, α was immobilized on a plate and then incubatedwith mixtures of β-clamp and wt TrfA in different molar ratios.The interaction of α with β-clamp was detected by β-specificantiserum (Fig. 5F). The measured levels of absorbance signalwere the same regardless of the increasing concentrations of thecompetitor protein TrfA in the mixtures. These data demon-strate that interaction between TrfA and α does not interferewith TrfA and α interactions with β. Indeed, tripartite complexesbetween TrfA, α, and β could be formed (Fig. 5D). The analyzedmultiprotein interactions are even more complex when consid-ering the dimeric form of β. The observed TrfA–α interaction

could explain how α becomes a part of the nucleoprotein com-plex at oriV in the presence of TrfA variants that are not able tointeract with and recruit the β-clamp.RK2 DNA replication is unidirectional and progresses down-

stream from the iterons. Presumably, this directionality is a conse-quence of how the replication complex is assembled. We consideredthe possibility that there was a factor(s) affecting strand specificityof primer synthesis and replisome assembly in RK2. Based on therecently published observations that TrfA interacts with ssDNAcontaining 13-mers of RK2 DUE (29), we hypothesized that TrfAcould be the factor determining replication directionality. Wetherefore decided to test if TrfA could contribute to strand-specificreplisome assembly.First, we reconstituted in vitro the general priming reactions on

three types of circular ssDNA templates. We prepared templatesBlueKS(−), oriV top, and oriV bottom with the use of pBluescriptKS(−) (Stratagene) (Fig. 6A and SI Materials and Methods). WhenDnaB, primase, Pol III core subunits, β-clamp, and clamp loader,but neither SSB nor TrfA, were present in the reaction, we detectedDNA synthesis on all three templates tested (Fig. 6 A and B).Under these conditions, primase was loaded nonspecifically andefficient DNA synthesis was observed. When SSB was added, thisgeneral priming reaction was limited (37) and we observed a sub-stantial reduction of DNA synthesis on all three templates tested;DNA synthesis was reduced from over 750 pmol to ∼140 pmol (Fig.6B). Under the experimental conditions used, we were not able to

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Fig. 4. Detection of α- and β- subunits on RK2 plasmid DNA in crude cell extract. Nucleoprotein complexes were formed with the use of an E. coli C600 crudecell extract, and gel filtration was carried out as described in SI Materials and Methods. Reaction mixtures, prepared as for in vitro replication (FII), containedpKD19L1 or non-oriV pUC18 (2.3 nM), wt TrfA (120 nM), TrfA F138A (120 nM), TrfA ΔLF (120 nM), or no TrfA (as indicated). After a 30-s incubation, reactionswere cross-linked by the addition of formaldehyde (0.01%) and incubated for an additional 10 min. Fractions collected (40 μL) during gel filtration wereapplied to an ELISA MaxiSorp plate (Nunc). (A–F) The presence of α or (G–I) βwas detected using an immune-enzymatic assay with anti–α- or anti–β-antibodies,respectively, and A at 450 nm was measured. Nucleoprotein complexes were eluted in void fractions (marked with gray). (Top) Presence of DNA was analyzedusing agarose electrophoresis.

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reduce this level of synthesis further. When reactions containingSSB were supplemented with ClpX-activated wt TrfA, DNA syn-thesis was restored up to 400 pmol on the ssDNA template con-taining oriV bottom strand but not when either the BlueKS(−) ororiV top ssDNA template was used (Fig. 6C). The TrfA variantsdefective in β-clamp interaction, TrfA F138A and TrfA ΔLF, werenot able to initiate DNA synthesis on the oriV bottom-strandtemplate (Fig. 6D). The reaction with wt TrfA required otherreplication proteins, including DnaB helicase, primase, Pol III core,γδδ′χψ, τ, and β-clamp (Fig. 6E). These results indicated that thereplisome complex was assembled on the bottom strand of oriV in aTrfA-dependent mode. The ssDNA template used consisted of theentire oriV bottom strand with iterons and DUE 13-mers. UsingSPR with a streptavidin matrix-coated (SA) sensor chip (SI Materialsand Methods), we found that wt TrfA, as well as TrfA F138A andTrfAΔLF, was able to interact with ssDNA containing the sequenceof the bottom-strand 13-mers but did not interact with ssDNAcontaining the top-strand 13-mers (Fig. S7 A–F). Under the sameexperimental conditions, none of the TrfA variants interacted withbottom-strand iterons and interaction with top-strand iterons wasvery limited (Fig. S7G–L). These SPR results, along with the resultsfrom our in vitro replication assays on ssDNA templates, demon-

strate that through its interaction with 13-mers, TrfA contributes tothe strand-specific assembly of the replication complex.

DiscussionOur results show that interaction of the RK2 plasmid Rep TrfAwith the β-clamp is essential for the formation of a fully activereplisome at the origin of supercoiled plasmid DNA. Althoughwe were able to demonstrate a TrfA and β-clamp protein–protein interaction, we failed to demonstrate a tripartite com-plex consisting of the β-clamp and TrfA bound to iterons con-taining a linear dsDNA fragment. Instead, we showed β-clampassociation with supercoiled plasmid DNA in the presence ofother factors, including TrfA. Our experiments with super-coiled dsDNA templates did not distinguish between β-clamploading on the leading and lagging strands. However, becausewe investigated origin-specific replication initiation completedwith de novo assembly of the replication complex, the detectionof the β-clamp complex on supercoiled DNA indicates the as-sembly process. β-Clamp complex formation on the templaterequires not only TrfA with an intact QLSLF motif, which isnecessary for the protein to interact with the β-clamp, but alsoother replication proteins, including DnaA, DnaB, primase,and clamp loader. The Pol III core, however, was not required,and the absence of gyrase reduced β-clamp complex formationonly slightly. The absence of DnaA in this complex results in aninefficient origin opening by TrfA (26); disturbances in helicaserecruitment (28); and, as we demonstrate in this work, defectsin β-clamp delivery on plasmid DNA. These observations sup-port an indirect involvement of DnaA in replisome assembly.However, we cannot exclude the possibility that DnaA, throughits contact with Hda protein (38), which is known to interactwith DNA-loaded β-clamp (39), has a more direct effect on theβ-clamp complex at the replication origin.DnaB helicase plays an important part in this nucleoprotein

puzzle. It contributes to replisome assembly through its in-teraction with τ (12) and primase (13), as well as by the ex-tension of an opened DUE. The extended ssDNA templateserves as the site for replisome assembly and activity. We havedemonstrated that the absence of DnaB results in a disturbancein β-clamp delivery on the plasmid DNA. Although helicaseprovides a structural scaffold for replisome assembly, primasemust be present as well. Primase synthesizes RNA primers thatare required for β-clamp and subsequent core subunit in-teraction with the primed DNA template (14). When nucleo-tides were omitted from reaction mixtures, we still detected aβ-clamp complex on supercoiled plasmid DNA (Fig. S5 A–D),which is most likely being due to primase using dNTPs (40, 41)or ATP (42) in the reaction mixture for primer synthesis. Dueto DnaB and DnaC requirements for ATP, we could not omitATP from reaction mixtures.We demonstrate that the clamp loader and TrfA with the

intact QLSLF motif for β-interaction are both indispensable forβ-clamp complex assembly on RK2 DNA. In our in vitro repli-cation assay, both τ and γ can assemble into functional clamploader; however, when τ was omitted, the level of DNA synthesiswas reduced (Fig. S6A). A similar effect was observed duringexperiments using ssDNA template (Fig. 6). Most likely, due tointeraction with α, τ provides stability of the clamp loader at thereplication fork or allows lagging strand synthesis when dsDNAis used as a template. It is intriguing to imagine the exact role ofTrfA in β-clamp complex formation at oriV. By performing anATPase assay, we excluded the possibility that TrfA affectsclamp loader ATPase activity (Fig. S6B). One possible mecha-nism for β-complex formation at oriV is that TrfA interacts withβ-clamp and hands off the β-clamp to the clamp loader followingprimer synthesis by primase. The clamp loader is responsible forβ-clamp opening and loading it onto the primed DNA. TheQLSLF motif in TrfA is located in the proximity of the WH

A

EB

C F

D

Fig. 5. TrfA interacts with α, and this interaction does not affect TrfA–β-clampcomplex formation. TrfA interactions with α and β-clamp were examined usingELISA (details are provided in SI Materials and Methods) (results presented fromn = 3 replicates, with the SD shown). (A–C) Graphs represent interactions be-tween individual proteins. (D) Possible network of interactions between TrfAand α and β-clamp. β-clamp binding pentapeptide motifs present in α (QADMF)and in TrfA (QLSLF) are marked. (E and F) Graphs represent competition ex-periments (details are provided in SI Materials and Methods). (E) TrfA wasimmobilized on the plate, and a mixture of β-clamp (250 nM) and α (increasingconcentration) was added. (F) α was immobilized on the plate, and a mixture ofβ-clamp (250 nM) and TrfA (increasing concentration) was added.

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domains responsible for the protein’s interaction with DNA (43).It is worth noting that changing the QLSLF motif into ALSMAalso results in a replicatively inactive TrfA (Fig. S8). Changing

the QTSMAF motif into ATSMAF in e-subunit of Pol III coreprevents interaction with the β-clamp (44). Experiments pre-sented in this work demonstrate that the F138A substitutionaffects TrfA interaction with the β-clamp but also somehow af-fects helicase activity at oriV (Fig. 2). It is difficult to speculate onthe nature of this dual effect. TrfA interacts with DnaB (27, 45);however, the interacting motifs have not been characterized. TheF138A substitution might have altered TrfA’s secondary struc-ture, thus influencing interaction with DnaB.We have revealed the interaction of TrfA with Pol III α-sub-

unit; however, the relevance of this interaction is not clear. Be-cause a nucleoprotein complex containing the β-clamp wasobserved even in the absence of core, the TrfA interaction with αmust not be critical to the assembly of β-clamp on oriV DNA.TrfA mutants in the QLSLF motif are able to interact with α,and in the presence of those mutants, the α-complex was ob-served on the plasmid DNA. It must be pointed out that TrfAΔLF, which is deficient in β-complex formation at oriV, does notpromote DNA synthesis. Those findings imply that β-loading isessential for proper assembly of active Pol III core at oriV. Wecould assume that TrfA interaction with α is required forα-recruitment, although other proteins (i.e., τ associated withDnaB) could also be involved in this process. The specific motif inTrfA responsible for its interaction with α needs to be identified.Because RK2 is a broad-host-range plasmid, it is of great im-portance to verify if the TrfA interacts with α-subunits of Pol IIIholoenzymes from other gram-negative bacteria.The experiments that used supercoiled dsDNA templates

demonstrate that TrfA protein–protein interactions contributeto the assembly of the replication complex at the plasmid origin,whereas the reconstitution of a strand-specific, 13-mer, andTrfA-dependent in vitro DNA replication reaction on ssDNAtemplates reveals how the direction of replication is determined.It is clear that assembly of replication machinery on the top orbottom, or on both the top and bottom, strands of melted DUEallows, respectively, unidirectional or bidirectional DNA repli-cation. It is not clear, however, what predisposes or determinesthe strand specificity of replisome assembly. It is assumed thathelicase/primase complex assembly and strand-specific primersynthesis are key factors in this process; however, before thiswork, it remained uncertain as to what determined the strandspecificity of helicase/primase loading. The reconstitution ofRK2 strand-specific DNA replication reactions on ssDNA tem-plates demonstrated that the TrfA interaction with DUE bot-tom-strand 13-mers and the protein’s interaction with thepolymerase β-clamp contribute to anchoring of the replisome onthe bottom strand of the plasmid origin. We were able to detectDNA synthesis only with wt TrfA, but not TrfA mutants, de-ficient in β-clamp interaction, and only with ssDNA templatescontaining the oriV bottom strand with 13-mer sequences. Ourdata indicated that the replisome complex was assembled in astrand-dependent and TrfA-dependent mode and requires DnaBhelicase and primase. We have previously shown that TrfA in-teracts with DnaB (45) and loads it onto oriV DUE (27). Whenusing ssDNA templates, TrfA is not required for DUE opening;rather, it is required for loading, the helicase/primase complex,and replisome assembly. In our experiments, a replication com-plex containing Pol III core for leading strand synthesis wasformed on the bottom strand, consistent with the previouslydetermined direction of RK2 plasmid replication (26, 46).It is of great interest to determine if a similar interaction network

exists in other plasmids. It would be of interest to determine if RK2as a broad-host-range replicon uses the same network of in-teractions in bacterial hosts other than E. coli. It is very likely thatthe recruitment and assembly of the replisome at the replicationorigin of an extrachromosomal genetic element requires specificmechanisms that differ from the mechanisms used at the bacterialchromosomal origin. However, Rep-dependent interactions with

A

B C

D E

Fig. 6. TrfA initiates DNA replication on bottom-strand oriV 13-mers. (A) Diagramof the in vitro-reconstituted DNA replication reactions whose results are shown inB–D. The reactions were performed with three types of ssDNA template. BlueKS(−)is ssDNA recovered from E. coli cells containing pBluescript KS(−), oriV top is ssDNAcontaining the sequence of RK2 oriV top strand, and oriV bottom is ssDNAcontaining the sequence of RK2 oriV bottom strand. The nonspecific generalpriming reaction by DnaB helicase and primase on all three templates is depictedat the top of the summary (top arrow). This reaction is inhibited by SSB. TrfA-dependent and template-specific ssDNA replication in the presence of SSB isdepicted at the bottom (bottom arrows). (B) SSB inhibits nonspecific DNA synthesison all three ssDNA templates. (C) SSB-coated oriV bottom ssDNA was replicated atincreasing concentrations of ClpX-activated wt TrfA, although neither oriV top norBlueKS(−) ssDNA templatewas replicated under the same experimental conditions.(D) Presence of ClpX-activated wt TrfA was essential for replication of SSB-coatedssDNA oriV bottom. TrfA variants defective in interaction with β-clamp did notinitiate DNA synthesis on oriV bottom-strand ssDNA template. (E) Requirementsfor DNA synthesis on SSB-coated ssDNA oriV bottom-strand template weredetermined using a protein omission approach.

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specific Pol III holoenzyme subunits and with DUE, as described inthis work, should be considered as an attractive model for strand-specific recruitment of replication machinery to any origin, andthereby determination of replication directionality.

Materials and MethodsA detailed list of plasmids and bacterial strains used in this study is presented inTable S1. Protocols and bacterial strains used for the purification of E. coligyrase and Pol III subunits were kindly provided by N. Dixon (University ofWollongong, Wollongong, Australia). Protocols and bacterial strains usedfor the purification of E. coli primase and HU were kindly provided byR. McMacken (Johns Hopkins University, Baltimore, MD). The protocol andbacterial strain used for the purification of the E. coli β-clamp were kindlyprovided by D. Bastia (Medical University of South Carolina, Charleston, SC).E. coli proteins DnaA-His6, DnaB-His6, DnaC, and ClpX were purified as de-scribed (43, 47–49). All TrfA preparations used in the tests were N-terminallyhistidine-tagged 33-kDa versions of TrfA. The wt TrfA, TrfA F138A, and TrfAΔLF were purified according to the method of Toukdarian et al. (33). E. coli

DNA Pol III subunits were a kind gift from M. O’Donnell, The RockefellerUniversity, New York, NY. Detailed information on the commercially availableproteins, antibodies, and reagents used in this study is given in SI Materialsand Methods.

An in vitro replication assay (FII) was performed as described by Kittell andHelinski (50), ClpX-dependent activation of TrfA was performed according tothe method of Pierechod et al. (43), and ELISA analyses were performed asdescribed previously (36), with details given in SI Materials and Methods. De-tailed protocols for in vivo replication activity, isolation of nucleoproteincomplexes by gel filtration, helicase activity assay (FI*), SPR, and GMSA analysisare given in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Prof. D. Bastia, Prof. N. Dixon, Prof.R. McMacken, and Prof. M. O’Donnell for providing bacterial strains and pro-teins. We thank Dr. A. Toukdarian for critical reading of the manuscript. Thiswork was supported by the Polish National Science Centre (Grant 2012/04/A/NZ1/00048) and the Foundation for Polish Science (Grant TEAM/2009-3/5). The openaccess publication cost was supported by the European Commission from the FP7Project Centre of Molecular Biotechnology for Healthy Life (MOBI4Health).

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