Plasmid Replication Initiator Interactions with Origin 13-Mers And

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Plasmid replication initiator interactions with origin PNAS PLUS 13-mers and polymerase subunits contribute to strand-specific replisome assembly

Aleksandra 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 been around the DNA with the use of the clamp loader (reviewed in extensively investigated, it is not clear what the exact mechanism ref. 18). The scenario for the polymerase assembly at the repli- for de novo assembly of the replication complex at the replication cation origin is mainly assumed, based on investigations of the origin is, or how the directionality of replication is determined. mechanism of leading and lagging DNA strand synthesis, con- Here, using the plasmid RK2 replicon, we analyze the protein in- ducted with in vitro assays on primed circular DNA but not on teractions required for Escherichia coli polymerase III (Pol III) ho- supercoiled templates. It is not clear how the replisome is as- loenzyme association at the replication origin. Our investigations sembled on supercoiled dsDNA after origin opening and revealed that in E. coli, replisome formation at the plasmid origin whether the helicase interactions with primase and τ-subunit are involves interactions of the RK2 plasmid replication initiation pro- the only factors contributing to de novo replisome assembly at tein (TrfA) with both the polymerase β-andα-subunits. In the the replication origin. presence of other replication proteins, including DnaA, helicase, In case of bacterial plasmids, involvement of both the plasmid- primase and the clamp loader, TrfA interaction with the β-clamp encoded Rep and the host-encoded replication initiator DnaA contributes to the formation of the β-clamp nucleoprotein complex was reported as essential for origin opening and helicase com- on origin DNA. By reconstituting in vitro the replication reaction on plex recruitment (19–21). DNA replication of the broad-host-

ssDNA templates, we demonstrate that TrfA interaction with the range plasmid RK2 (reviewed in ref. 19) is initiated by the RK2 BIOCHEMISTRY β-clamp and sequence-specific TrfA interaction with one strand of plasmid encoded Rep protein (TrfA), which binds to direct re- the plasmid origin DNA unwinding element (DUE) contribute to peats (iterons) localized at the plasmid’s replication origin (oriV) strand-specific replisome assembly. Wild-type TrfA, but not the (22) (Fig. 1A). In contrast to DnaA (23), TrfA, as well as other TrfA QLSLF mutant (which does not interact with the β-clamp), in plasmid Reps, does not contain a DNA binding domain (DBD). the presence of primase, helicase, Pol III core, clamp loader, and Instead, the plasmid Reps are similar to eukaryotic replication β-clamp initiates DNA synthesis on ssDNA template containing initiators and contain a winged helix (WH) domain for DNA 13-mers of the bottom strand, but not the top strand, of DUE. Re- interaction (24, reviewed in 25). TrfA interaction with the iterons sults presented in this work uncovered requirements for anchoring leads to origin opening assisted by host HU and DnaA proteins polymerase at the plasmid replication origin and bring insights of (26). TrfA plays a crucial role in DnaB helicase recruitment and how the directionality of DNA replication is determined. positioning at the AT-rich region of the oriV (27, 28). In contrast to DnaA protein, no data for plasmid Rep filament formation on DNA replication initiation | polymerase III | β-clamp | Rep | plasmid RK2 ssDNA have been provided to date. Recently, it was shown that TrfA interacts with ssDNA containing 13-mer sequences of one NA synthesis of prokaryotic and eukaryotic replicons re- Dquires the coordinated action of several enzymes (reviewed Significance in detail in 1, 2). These enzymes cooperate to form specific nucleoprotein complexes during the course of DNA replication. Research on DNA replication initiation has not revealed the ex- The formation of the initial complex is a result of a replication act mechanism for replication complex de novo assembly at the initiation protein (Rep) or origin recognition complex binding to ori origin or how the directionality of replication is determined. To dsDNA within the origin of DNA replication initiation ( ). This date, no evidence for direct involvement of a replication initia- interaction of the replication initiators with DNA results in ori- tion protein (Rep) in the process of polymerase recruitment has gin opening [i.e., destabilization of the DNA unwinding element been reported. This work demonstrates that a plasmid Rep, in (DUE)]. Origin opening provides ssDNA for helicase (3, 4), addition to its already described functions in origin opening and primase (5), and polymerase. helicase recruitment, can serve as a DNA polymerase anchoring It has been demonstrated that during the opening of the oriC factor. Through its interaction with 13-mer sequences on one bacterial chromosomal origin ( ), the chromosomal replica- strand of initially unwound DNA and interactions with the tion initiator, DnaA, binds to specific sequences (DnaA boxes) subunits of DNA polymerase, the initiation protein facilitates (6) and forms a filament on ssDNA (7, 8). Specific interaction strand-specific replisome assembly at the replication origin. This between DnaA and the DnaB helicase (9, 10) recruits the heli- step determines the direction of DNA replication. case and contributes to its loading by a helicase loader, the DnaC τ protein (11). Interactions between DnaB and the -subunit of Author contributions: I.K. designed research; A. Wawrzycka, M.G., and A. Wasaznik per- polymerase (12), as well as DnaB and primase (13), contribute to formed research; A. Wawrzycka, M.G., and I.K. analyzed data; and A. Wawrzycka and I.K. replisome assembly at Escherichia coli oriC. The primase re- wrote the paper. quires contact with single-stranded DNA-binding protein (SSB) The authors declare no conflict of interest. (14) to remain bound to the RNA primer. Disruption of this This article is a PNAS Direct Submission. interaction mediated by the polymerase clamp loader leads to Freely available online through the PNAS open access option. β primase displacement (14); -clamp loading on primed DNA 1To whom correspondence should be addressed. Email: [email protected]. (15, 16); and, finally, interaction of the polymerase core subunits edu.pl. β – β with the -clamp loaded template (14, 17). -clamp loading is a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complex reaction involving clamp opening and then positioning 1073/pnas.1504926112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1504926112 PNAS Early Edition | 1of9 Downloaded by guest on September 28, 2021 changed into Ala). Wild-type TrfA (wt TrfA) and TrfA variants A with alterations within the QLSLF motif were purified by affinity chromatography (Materials and Methods). To assess the quality of the purified proteins, we performed an analysis of the iso- thermal CD spectra (Fig. S1) and calculated the content of the respective secondary structures for each TrfA variant. The results did not reveal any substantial differences in the secondary BCstructure’s content of the analyzed TrfA variants in comparison to the wt TrfA. We then determined how the TrfA variants interacted with the β-clamp using surface plasmon resonance (SPR) (SI Materials and Methods and Fig. S2). The wt TrfA immobilized on a CM5 sensor chip interacted with the β-clamp, whereas TrfA ΔLF–β-clamp complex formation was severely impaired. Under the same experimental conditions, TrfA F138A interacted with the β-clamp, although slightly less efficiently than was observed for the wt TrfA. To determine whether the mutations in the QLSLF motif that altered TrfA’s interaction with the β-clamp influenced RK2 DNA synthesis, we tested the replication activity of the purified TrfA variants in an in vitro replication assay. The test was based on E. coli cell crude extract (FII) that allows replication of D supercoiled dsDNA in the presence of the plasmid replication initiator, TrfA. The soluble FII extract contains all proteins necessary for plasmid DNA synthesis, including polymerase III (Pol III) holoenzyme and chaperones for TrfA activation. DNA synthesis in this assay, measured as the total amount of incorporated nucleotides, reached a maximum when 90 nM wt TrfA was added to the assay mix and was inhibited by larger amounts of this protein (Fig. 1B). Based on the amount of nucleotides incorporated into the template, we calculated that 25% of DNA templates were typically copied, similar to results pre- Δ Fig. 1. TrfA LF is not active in RK2 DNA replication either in vitro or in sented by others during experiments with an oriC in vitro system vivo. (A) Plasmid RK2 minimal origin of replication. The scheme presents the (31, 32). TrfA ΔLF was defective in DNA synthesis; replication RK2 origin (oriV) region comprising the cluster of 17-bp direct repeats (iterons), four DnaA boxes, and the DUE region with four 13-mers. (B)In reactions carried out in the presence of varying amounts of this vitro replication with a crude extract (FII) prepared from E. coli C600. (C)In mutant protein remained at background levels. Replication ac- vitro DNA replication reaction reconstituted with purified proteins (Recon. tivity of the TrfA F138A mutant was reduced in comparison to system). (B and C) Both in vitro replication experiments were established wt TrfA but showed a similar activity profile with a peak at with increasing amounts of wt TrfA or TrfA mutants as noted (0, 30, 60, 90, 90 nM protein and an inhibition of DNA synthesis at larger 120, 150, 210, and 300 nM) (results are presented from n = 3 replicates, with amounts of protein. Similar results (Fig. 1C) were obtained when the SD shown). (D) TrfA in vivo activity test. Plasmid pSV16 containing oriV we performed in vitro replication assays using purified enzymes α was used to transform E. coli DH5 cells with plasmids carrying variants and a supercoiled dsDNA oriV template according to the method of trfA as indicated. The transformation frequency is reported as the of Konieczny and Helinski (28), with modifications (SI Materials number of colony-forming units per 1 μg of supercoiled plasmid pSV16 and Methods DNA averaged from three independent experiments, with the SD shown. ). The in vitro replication results were consistent with the replication results obtained in transformation efficiency tests. Plas- strand of the plasmid origin DUE (29). Interestingly, the specific mid pSV16 was used to transform E. coli cells containing pAT motif (QL[S/D]LF) determining interaction with the β-clamp has plasmid encoding one of the trfA gene variants: wt trfA, trfAΔLF, trfAF138A been identified in plasmid Reps (30); however, the relevance of or . Plasmid pSV16 contains the RK2 origin of repli- the interaction between the β-clamp and Reps has not been cation but not the gene for TrfA; thus, transformant colony growth indicates that the pAT encodes a replicatively active form established. The QAMSLF motif (related to QLSLF) was E. coli wt trfA identified in the δ-subunit of the clamp loader, and it has been of TrfA. When carrying pAT was transformed with shown to be required for δ-interaction with the β-clamp and pSV16 plasmid, colonies were obtained with a transformation frequency of 3.8 × 104 (Fig. 1D). No colonies were observed clamp loading (16). In this work, we investigate the significance when E. coli cells containing plasmid pATtrfAΔLF were trans- of the β-clamp interaction with the TrfA replication initiator in formed with pSV16. When E. coli containing pATtrfAF138A was the process of RK2 plasmid DNA replication initiation. We also transformed with pSV16, the obtained transformation frequency examine the requirements for the assembly of the replication was slightly lower compared with the transformation frequency complex at the plasmid origin. observed with pATwt trfA encoding wt TrfA. In a control ex- Results periment, when pBBR1MCS2 plasmid (Table S1), replicating independent of the TrfA, was used instead of pSV16, we ob- To address the question concerning the significance of TrfA served colony growth of E. coli strains with all three pAT variants β interaction with the -clamp, we constructed TrfA variants with tested (Fig. 1D). mutations in the QLSLF motif that were expected to disrupt this Our experiments revealed that besides the defects in TrfA interaction. With the use of an ELISA test, it was previously interaction with β-clamp, the analyzed mutations within the shown that LF deletion within the QLSLF motif results in TrfA QLSLF motif result in a deficiency of plasmid DNA synthesis defective in β-clamp binding (30). Using PCR-based site-directed both in vitro and in vivo. We then asked at which stage of rep- mutagenesis, we constructed plasmids carrying genes for TrfA lication initiation the ΔLF and F138A mutations caused the ΔLF and TrfA F138A (where Phe in the QLSLF motif was observed deficiencies of the in vitro and in vivo replication.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1504926112 Wawrzycka et al. Downloaded by guest on September 28, 2021 To answer this question, we examined the individual steps of A PNAS PLUS the initiation process in vitro. First, we tested if mutations within the QLSLF motif might affect TrfA interaction with DNA. TrfA binds to oriV iterons as a monomer (33), and chaperone proteins are required for TrfA activation by converting TrfA dimers to monomers (34, 35). Thus, before the DNA interaction tests, we used the E. coli ClpX chaperone to activate the purified TrfA variants (Materials and Methods). After incubation with the chaperone, we used a gel mobility shift assay (GMSA) (SI Ma- terials and Methods) to analyze binding of the ClpX-activated TrfA variants to fluorescently labeled dsDNA fragments containing a five-iteron sequence of RK2 oriV. Our experiments revealed no substantial differences among the tested proteins in their ability to form nucleoprotein complexes; TrfA ΔLF and TrfA F138A bind to the DNA fragment and produce retarded bands as efficiently as wt TrfA (Fig. S3 A–C). It must be pointed out that, as has been previously demonstrated, the ClpX does not interact with DNA (36), which we also verified during our experiments (Fig. S3D). B Because TrfA is involved in DnaB helicase complex formation at oriV (27, 28), we decided to test if the TrfA ΔLF and TrfA F138A, which were defective in replication, were able to recruit the helicase. Using gel filtration on a Sepharose CL-4B column (GE Healthcare), we tested helicase complex formation on a supercoiled DNA template containing oriV (pKD19L1) in the presence of ClpX-activated TrfA variants (Fig. 2A). In addition to supercoiled DNA, TrfA variants, and DnaB, the reaction

mixture contained DnaA and DnaC proteins, which are required Fig. 2. In the presence of TrfA ΔLF or TrfA F138A, the DnaB helicase com- BIOCHEMISTRY for helicase complex formation at oriV (28). Under these ex- plex is loaded and activated on RK2 DNA. (A) Formation of nucleoprotein perimental conditions, plasmid DNA was found in the column’s complexes was studied using gel filtration as described in SI Materials and void volume fractions (Fig. 2A, Top). Other panels of Fig. 2 Methods. Reaction mixtures containing each of the ClpX-activated TrfA illustrate DnaB detected by a protein-specific antiserum in variants (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 by consecutive fractions collected from the column. In a control μ experiment, when wt TrfA was not added to the reaction mix- gel filtration. Fractions (80 L) collected from the column were analyzed by SDS/PAGE and Western blotting for the presence of DnaB and by agarose ture, we did not detect DnaB in the void volume, which indicated oriV electrophoresis for the presence of DNA. (Top) Plasmid DNA was found in a lack of helicase at in the absence of TrfA. In contrast, void volume fractions. (B) E. coli DnaB helicase activity on RK2 DNA was DnaB protein was observed in void volume fractions when the determined by the FI* assay. The position of the FI* DNA band, indicating reaction mixture contained any of the tested variants of TrfA, helicase activity, is marked with arrows. Reaction mixtures were as described showing that the TrfA ΔLF supports helicase complex formation in SI Materials and Methods with increasing TrfA concentrations (0, 30, 60, on RK2 DNA as well as the wt TrfA (Fig. 2A). TrfA F138A 90, 120, and 150 nM in lanes 1–6, respectively). supports helicase complex formation less efficiently. In control experiments, we did not detect DnaB in void volume when pUC18 supercoiled DNA or no DNA was present in the reaction have to be accomplished in the presence of an active form of TrfA. Δ mixture (Fig. 2A). Our results indicate that TrfA LF, as well as TrfA F138A, pro- It has been demonstrated that in addition to DnaB loading, motes helicase activation but not plasmid DNA synthesis. These –β TrfA is involved in helicase activation at oriV (28). Thus, for- results, together with the confirmed TrfA -clamp interaction, mation of the DnaB helicase complex on DNA might not nec- strongly suggest a role for TrfA during the assembly of the repli- essarily result in helicase activity. We therefore tested the TrfA some complex at the plasmid replication origin. To further ex- ’ variants as helicase-activating factors. E. coli DnaB helicase ac- amine TrfA s role in replisome assembly, we studied the formation tivity on supercoiled RK2 plasmid DNA template was identified of nucleoprotein complexes on supercoiled RK2 plasmid DNA electrophoretically by the formation of an extensively unwound using gel filtration with a fluorescently labeled β-clamp (Fig. 3). As form of the supercoiled plasmid DNA, designated FI* (28). The in previous experiments, wt TrfA, TrfA F138A, or TrfA ΔLF was formation of the FI* form is a result of the combined activities of initially activated by incubation with ClpX. Reaction mixtures were plasmid and bacterial Reps, with the presence of an active form prepared as for in vitro replication assays using purified enzymes of TrfA being essential. ClpX activation of TrfA variants was (SI Materials and Methods). After incubation, the reaction mixtures performed as for the previous in vitro tests, and the other re- were applied on a Sepharose CL-4B column and the collected action components were then added. Experiments were carried fractions were analyzed for the presence of DNA and fluo- out with increasing concentrations of the TrfA variants (Fig. 2B, rescently labeled β-clamp. A fluorescent signal in the void vol- lanes 2–6; lane 1 is the reaction mixture without added TrfA). In ume fractions containing plasmid DNA would indicate that the agreement with previous results (34), in the presence of ClpX- β-clamp was found in a nucleoprotein complex. In the presence activated wt TrfA, we observed the FI* DNA band that indicates of wt TrfA, the fluorescent signal was detected in the void vol- helicase unwinding activity. The FI* form of DNA was also ob- ume fractions (Fig. 3A), whereas when the TrfA ΔLF variant was served in the presence of both TrfA mutants tested, TrfA ΔLF used, fluorescence remained at background levels in the void and TrfA F138A; however, TrfA F138A was slightly less efficient volume. The observed fluorescence was significantly reduced in helicase activation (Fig. 2B). when TrfA F138A was tested under the same conditions (Fig. To acquire helicase unwinding activity on plasmid DNA, a 3A). We estimated that when wt TrfA was present in the re- number of steps, including the formation of an initial complex, actions, ∼15% of the total amount of the β-clamp used for the origin opening, and helicase complex assembly and its activation, experiment was detected in DNA-containing fractions. It should

Wawrzycka et al. PNAS Early Edition | 3of9 Downloaded by guest on September 28, 2021 be pointed out that a similar efficiency was obtained with the and F). These results indicated that TrfA, with an intact QLSLF DnaA-dependent reaction on oriC-containing templates (Fig. motif, is specifically required for β-clamp complex formation at S4). We also performed control experiments where reaction the RK2 plasmid origin. We then determined what other pro- mixtures contained only fluorescently labeled β-clamp and RK2 teins in the reaction mixture were indispensable for the β-clamp plasmid DNA or all components except TrfA. In both cases, the complex formation with supercoiled RK2 plasmid DNA. A fluorescence signal did not appear in void fractions (Fig. S5 E fluorescent signal was not detected in void fractions in the absence of all of the other Pol III subunits (Fig. 3B), γδδ′χψ (Fig. 3D), DnaA (Fig. 3E), DnaB (Fig. 3F), or primase (Fig. 3G). The presence of Pol III core subunits (Fig. 3C) or gyrase (Fig. 3H), however, was not essential for β-clamp nucleoprotein complex formation on RK2 plasmid DNA, because we still detected A E fluorescent signal in the void volume in the absence of either of these factors. When GTP, UTP, and CTP and/or dNTPs were omitted from the reaction mixture, we still observed a fluorescent signal in the void fractions (Fig. S5 B–D). Because β-clamp nucleoprotein complex formation was dependent on TrfA and γδδ′χψ in our gel filtration experiments, we tested how the composition of clamp loader affects the RK2 DNA replication. In vitro replication assays in a reconstituted system with variously assembled clamp loaders were performed (Fig. S6A). The reactions were performed with wt TrfA and TrfA ΔLF. A sub- B F stantial reduction of the replication activity was observed in the absence of τ-subunit, although not much difference in replication activity was observed when the reaction was conducted as in Fig. 1C, containing both τ and γ, in comparison to the reaction with clamp loader lacking γ (Fig. S6A). With TrfA ΔLF or without TrfA at all, we detected only a background level of DNA synthesis. To test if TrfA can affect ATPase hydrolysis by a clamp loader, we performed a clamp loader ATPase activity assay in the presence of primed DNA (Fig. S6B). No influence of wt TrfA or TrfA ΔLF was observed. To analyze the mechanism of replisome assembly at the RK2 CGplasmid origin further, we coupled the gel filtration experiment with immunodetection of the α-subunit of the Pol III core (Fig. 4). After incubation, reaction mixtures consisting of bacterial crude extracts (FII), RK2 supercoiled DNA, and TrfA variants were applied on a Sepharose CL-4B column and fractions were immunoanalyzed. The α wasdetectedinvoidvolumefractions when wt TrfA (Fig. 4A) or, surprisingly, the TrfA F138A or TrfA ΔLF mutant (Fig. 4 B and C) was present in reaction mixtures. In control experiments, when TrfA or DNA was omitted or pUC18 was used instead of RK2, α was not detected DHimmunologically in the void volume (Fig. 4 D–F). When we tested for β-clamp nucleoprotein complex formation under the same experimental conditions, the protein was detected in the presence of wt TrfA and oriV DNA (Fig. 4G). When TrfA was omitted or TrfA ΔLF was present in the incubation mixture, we observed almost no absorbance signal derived from immuno- logical assay. The observed absorbance signal was significantly reduced when TrfA F138A was used (Fig. 4 G–I). The β-clamp tracking in crude extract was in accordance with experiments performed for reconstituted reactions (Fig. 3A). Also, when we used a reconstituted system for α-tracking, consistent with the results obtained in crude extract, α was eluted in void fractions in the Fig. 3. TrfA is required for β-clamp nucleoprotein complex formation on RK2 presence of either wt TrfA or TrfA ΔLF (Fig. S4 D and E). Re- plasmid DNA. Nucleoprotein complex formation in the reconstituted system was α studied using a gel filtration-based assay. (A) Reaction mixtures containing wt gardless of the system used, the was found in the nucleoprotein TrfA (120 nM), TrfA F138A (120 nM), or TrfA ΔLF (120 nM) were activated with complex formed on RK2 DNA in the presence of the TrfA variants ClpX (870 nM), and DnaA (450 nM), DnaB (200 nM), DnaC (1.1 μM), gyrase defective in interaction with the β-clamp (Fig. S2)andnotpro- (13 nM), HU (110 nM), SSB (150 nM), primase (60 nM), and pKD19L1 (2.3 nM) moting the β-clamp complex formation at the plasmid origin. This template DNA were then added. (B–H) Reaction mixtures were prepared as unexpected finding raised a question concerning the mechanism of described for A, except that indicated components were omitted. A 20-min in- this interaction. cubation was carried out to form a helicase complex, and β-clamp labeled with To address the issue of how α interacts at oriV without the τ αeθτ Alexa 488 fluorescent dye (17 nM) and E. coli Pol III core -subunits [ (19 nM) β-clamp being loaded onto the plasmid DNA, we performed an and γδδ′χψ (2.5 nM)] were then added. After 15 s, the reaction was quenched by α β SI Materials and adding 5 units of hexokinase to remove ATP (51), followed by gel filtration on a ELISA with -specific or -specific antisera ( μ Methods and Fig. S1 C and D). Interestingly, when wt TrfA was Sepharose CL-4B column. The collected fractions (40 L) were analyzed for the α presence of the Alexa 488 β-clamp by measuring fluorescence and for the immobilized on plates and increased concentrations of were presence of DNA by agarose electrophoresis. (Top) Plasmid DNA was found in applied, we clearly detected an interaction (Fig. 5A). Furthermore, void volume fractions only (as labeled). TrfA ΔLF and TrfA F138A interacted with α as efficiently as wt

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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 crude cell extract, and gel filtration was carried out as described in SI Materials and Methods. Reaction mixtures, prepared as for in vitro replication (FII), contained pKD19L1 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, reactions were cross-linked by the addition of formaldehyde (0.01%) and incubated for an additional 10 min. Fractions collected (40 μL) during gel filtration were applied to an ELISA MaxiSorp plate (Nunc). (A–F) The presence of α or (G–I) β was 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 analyzed using agarose electrophoresis.

TrfA (Fig. 5A). Consistent with our SPR analysis (Fig. S2), we could explain how α becomes a part of the nucleoprotein com- observed that wt TrfA interacts with the β-clamp, whereas TrfA plex at oriV in the presence of TrfA variants that are not able to ΔLF is deficient in β-clamp binding (Fig. 5B) and TrfA F138A interact with and recruit the β-clamp. interaction with the β-clamp was reduced by half compared with RK2 DNA replication is unidirectional and progresses down- wt TrfA (Fig. 5B). We were also able to detect an α–β-clamp in- stream from the iterons. Presumably, this directionality is a conse- teraction (Fig. 5C). quence of how the replication complex is assembled. We considered These experiments revealed a network of interactions between the possibility that there was a factor(s) affecting strand specificity TrfA, β-clamp, and α (Fig. 5D). Interestingly, a β-clamp binding of primer synthesis and replisome assembly in RK2. Based on the motif related to the one found in TrfA is also present in α.To recently published observations that TrfA interacts with ssDNA determine if the identified interactions were competitive, we containing 13-mers of RK2 DUE (29), we hypothesized that TrfA performed two types of ELISA tests. In the first one, wt TrfA could be the factor determining replication directionality. We was immobilized on the plate and incubated with mixtures therefore decided to test if TrfA could contribute to strand-specific composed of β-clamp and α in different molar ratios (Fig. 5E). replisome assembly. By using β-specific antiserum, we detected a TrfA–β-clamp First, we reconstituted in vitro the general priming reactions on complex even when there was an excess of α. For the second three types of circular ssDNA templates. We prepared templates ELISA test, α was immobilized on a plate and then incubated BlueKS(−), oriV top, and oriV bottom with the use of pBluescript with mixtures of β-clamp and wt TrfA in different molar ratios. KS(−) (Stratagene) (Fig. 6A and SI Materials and Methods). When The interaction of α with β-clamp was detected by β-specific DnaB, primase, Pol III core subunits, β-clamp, and clamp loader, antiserum (Fig. 5F). The measured levels of absorbance signal but neither SSB nor TrfA, were present in the reaction, we detected were the same regardless of the increasing concentrations of the DNA synthesis on all three templates tested (Fig. 6 A and B). competitor protein TrfA in the mixtures. These data demon- Under these conditions, primase was loaded nonspecifically and strate that interaction between TrfA and α does not interfere efficient DNA synthesis was observed. When SSB was added, this with TrfA and α interactions with β. Indeed, tripartite complexes general priming reaction was limited (37) and we observed a sub- between TrfA, α, and β could be formed (Fig. 5D). The analyzed stantial reduction of DNA synthesis on all three templates tested; multiprotein interactions are even more complex when consid- DNA synthesis was reduced from over 750 pmol to ∼140 pmol (Fig. ering the dimeric form of β. The observed TrfA–α interaction 6B). Under the experimental conditions used, we were not able to

Wawrzycka et al. PNAS Early Edition | 5of9 Downloaded by guest on September 28, 2021 strate that through its interaction with 13-mers, TrfA contributes to the strand-specific assembly of the replication complex. A D Discussion Our results show that interaction of the RK2 plasmid Rep TrfA with the β-clamp is essential for the formation of a fully active replisome at the origin of supercoiled plasmid DNA. Although we were able to demonstrate a TrfA and β-clamp protein– protein interaction, we failed to demonstrate a tripartite complex consisting of the β-clamp and TrfA bound to iterons containing a linear dsDNA fragment. Instead, we showed β-clamp association with supercoiled plasmid DNA in the presence of other factors, including TrfA. Our experiments with super- B E coiled dsDNA templates did not distinguish between β-clamp loading on the leading and lagging strands. However, because we investigated origin-specific replication initiation completed with de novo assembly of the replication complex, the detection of the β-clamp complex on supercoiled DNA indicates the assembly process. β-Clamp complex formation on the template requiresnotonlyTrfAwithanintactQLSLFmotif,whichis necessary for the protein to interact with the β-clamp, but also other replication proteins, including DnaA, DnaB, primase, and clamp loader. The Pol III core, however, was not required, β CFand the absence of gyrase reduced -clamp complex formation only slightly. The absence of DnaA in this complex results in an inefficient origin opening by TrfA (26); disturbances in helicase recruitment (28); and, as we demonstrate in this work, defects in 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, through its contact with Hda protein (38), which is known to interact with DNA-loaded β-clamp (39), has a more direct effect on the β-clamp complex at the replication origin. Fig. 5. TrfA interacts with α, and this interaction does not affect TrfA–β-clamp DnaB helicase plays an important part in this nucleoprotein complex formation. TrfA interactions with α and β-clamp were examined using puzzle. It contributes to replisome assembly through its in- ELISA (details are provided in SI Materials and Methods) (results presented from teraction with τ (12) and primase (13), as well as by the ex- n = 3 replicates, with the SD shown). (A–C) Graphs represent interactions be- tensionofanopenedDUE.TheextendedssDNAtemplate tween individual proteins. (D) Possible network of interactions between TrfA serves as the site for replisome assembly and activity. We have and α and β-clamp. β-clamp binding pentapeptide motifs present in α (QADMF) demonstrated that the absence of DnaB results in a disturbance and in TrfA (QLSLF) are marked. (E and F) Graphs represent competition ex- in β-clamp delivery on the plasmid DNA. Although helicase periments (details are provided in SI Materials and Methods). (E) TrfA was provides a structural scaffold for replisome assembly, primase immobilized on the plate, and a mixture of β-clamp (250 nM) and α (increasing α must be present as well. Primase synthesizes RNA primers that concentration) was added. (F) was immobilized on the plate, and a mixture of β β-clamp (250 nM) and TrfA (increasing concentration) was added. are required for -clamp and subsequent core subunit interaction with the primed DNA template (14). When nucleotides were omitted from reaction mixtures, we still detected a reduce this level of synthesis further. When reactions containing β-clamp complex on supercoiled plasmid DNA (Fig. S5 A–D), SSB were supplemented with ClpX-activated wt TrfA, DNA syn- which is most likely being due to primase using dNTPs (40, 41) thesis was restored up to 400 pmol on the ssDNA template con- or ATP (42) in the reaction mixture for primer synthesis. Due taining oriV bottom strand but not when either the BlueKS(−)or to DnaB and DnaC requirements for ATP, we could not omit oriV top ssDNA template was used (Fig. 6C). The TrfA variants ATP from reaction mixtures. defective in β-clamp interaction, TrfA F138A and TrfA ΔLF, were We demonstrate that the clamp loader and TrfA with the oriV intact QLSLF motif for β-interaction are both indispensable for not able to initiate DNA synthesis on the bottom-strand β template (Fig. 6D). The reaction with wt TrfA required other -clamp complex assembly on RK2 DNA. In our in vitro replication assay, both τ and γ can assemble into functional clamp replication proteins, including DnaB helicase, primase, Pol III core, loader; however, when τ was omitted, the level of DNA synthesis γδδ′χψ, τ,andβ-clamp (Fig. 6E). These results indicated that the was reduced (Fig. S6A). A similar effect was observed during replisome complex was assembled on the bottom strand of oriV in a experiments using ssDNA template (Fig. 6). Most likely, due to TrfA-dependent mode. The ssDNA template used consisted of the α τ oriV interaction with , provides stability of the clamp loader at the entire bottom strand with iterons and DUE 13-mers. Using replication fork or allows lagging strand synthesis when dsDNA SI Materials SPR with a streptavidin matrix-coated (SA) sensor chip ( is used as a template. It is intriguing to imagine the exact role of and Methods ), we found that wt TrfA, as well as TrfA F138A and TrfA in β-clamp complex formation at oriV. By performing an Δ TrfA LF, was able to interact with ssDNA containing the sequence ATPase assay, we excluded the possibility that TrfA affects of the bottom-strand 13-mers but did not interact with ssDNA clamp loader ATPase activity (Fig. S6B). One possible mecha- containing the top-strand 13-mers (Fig. S7 A–F). Under the same nism for β-complex formation at oriV is that TrfA interacts with experimental conditions, none of the TrfA variants interacted with β-clamp and hands off the β-clamp to the clamp loader following bottom-strand iterons and interaction with top-strand iterons was primer synthesis by primase. The clamp loader is responsible for very limited (Fig. S7 G–L). These SPR results, along with the results β-clamp opening and loading it onto the primed DNA. The from our in vitro replication assays on ssDNA templates, demon- QLSLF motif in TrfA is located in the proximity of the WH

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1504926112 Wawrzycka et al. Downloaded by guest on September 28, 2021 A the QTSMAF motif into ATSMAF in e-subunit of Pol III core PNAS PLUS prevents interaction with the β-clamp (44). Experiments presented in this work demonstrate that the F138A substitution affects TrfA interaction with the β-clamp but also somehow affects helicase activity at oriV (Fig. 2). It is difficult to speculate on the nature of this dual effect. TrfA interacts with DnaB (27, 45); however, the interacting motifs have not been characterized. The F138A substitution might have altered TrfA’s secondary structure, thus influencing interaction with DnaB. We have revealed the interaction of TrfA with Pol III α-subunit; however, the relevance of this interaction is not clear. Be- cause a nucleoprotein complex containing the β-clamp was observed even in the absence of core, the TrfA interaction with α must 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 observed on the plasmid DNA. It must be pointed out that TrfA ΔLF, which is deficient in β-complex formation at oriV, does not promote DNA synthesis. Those findings imply that β-loading is essential for proper assembly of active Pol III core at oriV.We could assume that TrfA interaction with α is required for α-recruitment, although other proteins (i.e., τ associated with DnaB) could also be involved in this process. The specific motif in α BCTrfA responsible for its interaction with 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 III holoenzymes from other gram-negative bacteria.

The experiments that used supercoiled dsDNA templates BIOCHEMISTRY demonstrate that TrfA protein–protein interactions contribute to the assembly of the replication complex at the plasmid origin, whereas the reconstitution of a strand-specific, 13-mer, and TrfA-dependent in vitro DNA replication reaction on ssDNA templates reveals how the direction of replication is determined. It is clear that assembly of replication machinery on the top or DE bottom, or on both the top and bottom, strands of melted DUE allows, respectively, unidirectional or bidirectional DNA replication. It is not clear, however, what predisposes or determines the strand specificity of replisome assembly. It is assumed that helicase/primase complex assembly and strand-specific primer synthesis are key factors in this process; however, before this work, it remained uncertain as to what determined the strand specificity of helicase/primase loading. The reconstitution of RK2 strand-specific DNA replication reactions on ssDNA templates demonstrated that the TrfA interaction with DUE bottom-strand 13-mers and the protein’s interaction with the polymerase β-clamp contribute to anchoring of the replisome on Fig. 6. TrfA initiates DNA replication on bottom-strand oriV 13-mers. (A)Diagram the bottom strand of the plasmid origin. We were able to detect of the in vitro-reconstituted DNA replication reactions whose results are shown in DNA synthesis only with wt TrfA, but not TrfA mutants, de- B–D. The reactions were performed with three types of ssDNA template. BlueKS(−) ficient in β-clamp interaction, and only with ssDNA templates is ssDNA recovered from E. coli cells containing pBluescript KS(−), oriV top is ssDNA containing the oriV bottom strand with 13-mer sequences. Our containing the sequence of RK2 oriV top strand, and oriV bottom is ssDNA data indicated that the replisome complex was assembled in a containing the sequence of RK2 oriV bottom strand. The nonspecific general strand-dependent and TrfA-dependent mode and requires DnaB priming reaction by DnaB helicase and primase on all three templates is depicted helicase and primase. We have previously shown that TrfA in- at the top of the summary (top arrow). This reaction is inhibited by SSB. TrfA- teracts with DnaB (45) and loads it onto oriV DUE (27). When dependent and template-specific ssDNA replication in the presence of SSB is depicted at the bottom (bottom arrows). (B) SSB inhibits nonspecific DNA synthesis using ssDNA templates, TrfA is not required for DUE opening; on all three ssDNA templates. (C)SSB-coatedoriV bottom ssDNA was replicated at rather, it is required for loading, the helicase/primase complex, increasing concentrations of ClpX-activated wt TrfA, although neither oriV top nor and replisome assembly. In our experiments, a replication com- BlueKS(−) ssDNA template was replicated under the same experimental conditions. plex containing Pol III core for leading strand synthesis was (D) Presence of ClpX-activated wt TrfA was essential for replication of SSB-coated formed on the bottom strand, consistent with the previously ssDNA oriV bottom. TrfA variants defective in interaction with β-clamp did not determined direction of RK2 plasmid replication (26, 46). initiate DNA synthesis on oriV bottom-strand ssDNA template. (E) Requirements It is of great interest to determine if a similar interaction network for DNA synthesis on SSB-coated ssDNA oriV bottom-strand template were exists in other plasmids. It would be of interest to determine if RK2 determined using a protein omission approach. as a broad-host-range replicon uses the same network of interactions in bacterial hosts other than E. coli. It is very likely that the recruitment and assembly of the replisome at the replication domains responsible for the protein’s interaction with DNA (43). origin of an extrachromosomal genetic element requires specific It is worth noting that changing the QLSLF motif into ALSMA mechanisms that differ from the mechanisms used at the bacterial also results in a replicatively inactive TrfA (Fig. S8). Changing chromosomal origin. However, Rep-dependent interactions with

Wawrzycka et al. PNAS Early Edition | 7of9 Downloaded by guest on September 28, 2021 specific Pol III holoenzyme subunits and with DUE, as described in DNA Pol III subunits were a kind gift from M. O’Donnell, The Rockefeller this work, should be considered as an attractive model for strand- University, New York, NY. Detailed information on the commercially available specific recruitment of replication machinery to any origin, and proteins, antibodies, and reagents used in this study is given in SI Materials thereby determination of replication directionality. and Methods. An in vitro replication assay (FII) was performed as described by Kittell and Materials and Methods Helinski (50), ClpX-dependent activation of TrfA was performed according to the method of Pierechod et al. (43), and ELISA analyses were performed as A detailed list of plasmids and bacterial strains used in this study is presented in described previously (36), with details given in SI Materials and Methods.De- Table S1. Protocols and bacterial strains used for the purification of E. coli tailed protocols for in vivo replication activity, isolation of nucleoprotein gyrase and Pol III subunits were kindly provided by N. Dixon (University of complexes by gel filtration, helicase activity assay (FI*), SPR, and GMSA analysis Wollongong, Wollongong, Australia). Protocols and bacterial strains used are given in SI Materials and Methods. for the purification of E. coli primase and HU were kindly provided by R. McMacken (Johns Hopkins University, Baltimore, MD). The protocol and bacterial strain used for the purification of the E. coli β-clamp were kindly ACKNOWLEDGMENTS. We thank Prof. D. Bastia, Prof. N. Dixon, Prof. R. McMacken, and Prof. M. O’Donnell for providing bacterial strains and pro- provided by D. Bastia (Medical University of South Carolina, Charleston, SC). teins. We thank Dr. A. Toukdarian for critical reading of the manuscript. This E. coli proteins DnaA-His6, DnaB-His6, DnaC, and ClpX were purified as de- work was supported by the Polish National Science Centre (Grant 2012/04/A/NZ1/ – scribed (43, 47 49). All TrfA preparations used in the tests were N-terminally 00048) and the Foundation for Polish Science (Grant TEAM/2009-3/5). The open histidine-tagged 33-kDa versions of TrfA. The wt TrfA, TrfA F138A, and TrfA access publication cost was supported by the European Commission from the FP7 ΔLF were purified according to the method of Toukdarian et al. (33). E. coli Project Centre of Molecular Biotechnology for Healthy Life (MOBI4Health).

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