Gasset-Rosa. 2008 .MolMicrob

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Negative regulation of pPS10 plasmid replication: originpairing by zipping-up DNA-bound RepA monomers

Ftima Gasset-Rosa,1 Teresa Daz-Lpez,1

Rudi Lurz,2 Alicia Prieto,1

M. Elena Fernndez-Tresguerres1 and

Rafael Giraldo1*1Department of Molecular Microbiology, Centro de

Investigaciones Biolgicas CSIC, Madrid, Spain.2Max Planck Institute for Molecular Genetics, Berlin,

Germany.

Summary

In many plasmid replicons of Gram-negative bacteria,

Rep protein dimers are transcriptional self-repressors

of their genes, whereas monomers are initiators of

DNA replication. Switching between both functions

implies conformational remodelling of Rep, and is

promoted by Rep binding to the origin DNA repeats

(iterons) or chaperones. Rep proteins play another

key role: they bridge together two iteron DNA

stretches, found either on the same or on different

plasmid molecules. These so-called, respectively,

looped and handcuffed complexes are thought to

be negative regulators of plasmid replication.

Although evidence for Rep-dependent plasmid hand-cuffing has been found in a number of replicons,

the structure of these RepDNA assemblies is still

unknown. Here, by a combination of proteomics, elec-

tron microscopy, genetic analysis and modelling, we

provide insight on a possible three-dimensional

structure for two handcuffed arrays of the iterons

found at the origin of pPS10 replicon. These are

brought together in parallel register by zipping-up

DNA-bound RepA monomers. We also present evi-

dence for a distinct role of RepA dimers in DNA

looping. This work defines a new regulatory interface

in Rep proteins.

Introduction

Bacterial plasmids are extrachromosomal genetic ele-ments that control DNA replication to keep their copynumber within precise limits, characteristic for each

replicon. This control is achieved by a variety of molecularmechanisms, ranging from antisense RNA and transcrip-tional self-repression of the initiator gene (rep) to initiatorprotein titration (for comprehensive reviews see delSolar et al., 1998; Chattoraj, 2000; Krger et al., 2004;Paulsson and Chattoraj, 2006). In Gram-negative bac-teria, plasmid replicons such as P1, RK2, R6K, F andpPS10 (Fig. 1A), which contain directly repeatedsequences, known as iterons, at their replication origins(ori), have been extensively characterized. Rep proteinsundergo structural remodelling from being dimeric tran-

scriptional repressors to become monomeric initiators ofreplication. Initiators bind to the iterons and establish anucleoprotein complex that brings the bacterial hostfactors required for DNA replication to the ori(reviewed indel Solar et al., 1998; Giraldo and Fernndez-Tresguerres, 2004; Krger et al., 2004).

A common finding in iteron-containing plasmids isthe ability of Rep proteins to bridge together two arrays ofiteron DNA repeats. These can be located either on thesame plasmid molecule, with distal increpeats in cis(thusresulting in a DNA loop), or on different plasmid mol-ecules, in such a way that the ori/inc repeats in trans of

two plasmid circles become handcuffed. Rep-mediatedorigin handcuffing was initially described for P1 (Pal andChattoraj, 1988) and R6K (McEachern et al., 1989) repli-cons and is thought to be the basis for the incompatibilityphenomenon, in which two distinct plasmids carrying thesame replicon cannot stably coexist in a given cell in theabsence of selective pressure (Tolun and Helinski, 1981).Looping and handcuffing are believed to exert a sterichindrance on plasmid origins that prevents new rounds ofreplication initiation (Park et al., 2001) by inhibiting originmelting (Zzaman and Bastia, 2005). Such regulatorynucleoprotein complexes persist until the Rep assemblybridging iteron repeats is dismantled, most likely by dilu-tion upon cell growth (Das and Chattoraj, 2004) or chap-erone action (Uga et al., 1999).

Looping and handcuffing have been studied byapproaches such as Rep-enhanced self-ligation ofiterons (Miron et al., 1992; Mukhopadhyay et al., 1994;Uga et al., 1999; Das and Chattoraj, 2004; Das et al.,2005; Kunnimalaiyaan et al., 2005), monitoring the effectof Rep-dependent looping on DNA replication in vitro(Zzaman and Bastia, 2005) and analysing their function

Accepted 8 February, 2008. *For correspondence. E-mail [email protected]; Tel. (+34) 91 8373112; Fax (+34) 91 5360432.

Molecular Microbiology (2008) 68(3), 560572 doi:10.1111/j.1365-2958.2008.06166.xFirst published online 20 March 2008

2008 The AuthorsJournal compilation 2008 Blackwell Publishing Ltd


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by mathematical modelling (Morrison and Chattoraj,2004). It has been shown that isolated rep mutantsexhibit, to different extents, deficiencies in Rep-mediatedorigin coupling and thus a plasmid copy-up phenotype(Miron et al., 1994; Mukhopadhyay et al., 1994; Ugaet al., 1999). A major role for Rep dimers (rather than formonomers) in mediating origin pairing was initially pro-posed for the replication protein of R6K plasmid (Urhet al., 1998; Kunnimalaiyaan et al., 2005). Indirect evi-dence comes from the fact of handcuffing counteracting

by molecular chaperones (DnaK-J/GrpE triad), which wasattributed to an increase in the ratio of Rep monomers todimers (Das and Chattoraj, 2004; Zzaman and Bastia,2005). In the same sense it can be interpreted the inabilityof an excess of a monomeric Rep mutant, unlike thedimeric WT protein, to inhibit F plasmid replication in vitroin an assay dependent on Rep binding to increpeats in cis(Zzaman and Bastia, 2005). However, it is noteworthy thatRep initiator monomers are the molecular species with the

highest affinity for the iteron repeats (Papp et al., 1994;Giraldo et al., 1998; Urh et al., 1998), and in pPS10iterons even have an active role in displacing the equilib-rium between Rep dimers and monomers towards thelatter (Daz-Lpez et al., 2003). Therefore, it was pro-posed that dimers of the Rep protein of RK2 plasmidcould be bridging two monomer-bound iteron arrays(Toukdarian and Helinski, 1998), a model then extendedto handcuffing in F plasmid (Zzaman and Bastia, 2005).Three different interaction surfaces may exist in Rep pro-

teins for: (i) dimerization, (ii) head-to-tail intermonomercontacts (relevant for cooperative binding to the iteronrepeats) and (iii) handcuffing, which was first proposed onthe grounds of the crystal structure of the dimerizationdomain of RepA, the initiator protein of the pPS10 plasmid(Giraldo et al., 2003; Giraldo and Fernndez-Tresguerres,2004), and then reformulated because of the crystal struc-ture of a monomeric, multiple mutant of the Rep initiator ofthe R6K plasmid (Swan et al., 2006).

Fig. 1. A. Basic replicon of pPS10 plasmid (Nieto et al., 1992; Giraldo and Fernndez-Tresguerres, 2004). RepA protein dimers bind to aninverted repeat operator sequence (green arrows) to self-repress the transcription of repA gene. The sequence recognized by RepAC-terminal WH2 domain at the operator is also found at one half of each of four directly repeated sequences (iterons, magenta arrows) at theorigin region (oriV). Upon dissociation, the N-terminal WH1 domain experiences a conformational transformation (cyan squares blue ovals)that enables it to bind to the half of each iteron that remained empty.B. Three alternative models for RepA-mediated origin pairing (dubbed handcuffing), an assembly with a major role in controlling plasmidreplication. Top: WH2 domain from each dimer binds to a different plasmid molecule (Urh et al., 1998; Kunnimalaiyaan et al., 2005). Middle:Direct interactions between two arrays of RepA monomers bound to iterons in different plasmid molecules (Uga et al., 1999). Bottom: RepAdimers as staples that bridge together monomer-bound iterons (Toukdarian and Helinski, 1998; Das and Chattoraj, 2004; Zzaman and Bastia,2005). This model could be considered a combination of the former two. In the first and last cases, the dimerization interface in RepA wouldbe directly responsible for protein-protein contacts, whereas the middle and bottom models imply new interprotein contacts. If the iteron arrayswere found in the same plasmid molecule ( in cis), looping would have been generated instead of handcuffing ( in trans).

Structure ofhandcuffed RepAorigin DNA complexes 561

2008 The AuthorsJournal compilation 2008 Blackwell Publishing Ltd, Molecular Microbiology, 68, 560572


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A scheme of the various molecular models proposedfor handcuffing, involving either Rep dimers, monomers orboth, is shown in Fig. 1B, in which a parallel orientation ofthe two linked origin arrays is depicted, as reported for P1(Das and Chattoraj, 2004) and R6K (Urh et al., 1998)iterons. However, definitive evidence for one or the othermodel has not yet been provided for the regulatoryassemblies in plasmid replication (Das et al., 2005), suchas the molecular structure of a handcuffed Repiteroncomplex. This structural determination is a complex issue,because handcuffed iterons are unlikely to be crystallized,owing to their intrinsic heterogeneity and tendency toaggregate.

This study extends our knowledge on the RepAprotein of the Pseudomonas pPS10 replicon (reviewedin Giraldo and Fernndez-Tresguerres, 2004). Here wereport an integrated analysis of the higher order RepDNA complexes relevant for rep transcriptional repres-sion (RepAoperator complex) and handcuffing (twinnedRepAiteron complexes). We used transmission electron

microscopy (TEM) to visualize both types of RepADNAcomplexes and found that the orientation adopted bytwo handcuffed iteron arrays is parallel. By means of aproteomic approach to RepADNA complexes, includingprotein cross-linking, trypsin digestion and mass spec-trometry, we identified regions in RepA that becomeclose together upon protein assembly at the operator orthe iterons. By means of random mutagenesis, we alsoisolated RepA mutants affected in pPS10 incompatibility.These mutants remained dimeric, their DNA bindingability was unaltered, and they exhibited a plasmidcopy-up phenotype. They also showed a significant

reduction in handcuffing by TEM. A point mutantdesigned to affect intermonomer contacts but with noeffect on RepA dimerization exhibits the same pheno-type. Location of both the cross-linked residues and themutations in 3D models for the RepA dimers (dRepA)and for two monomers assembled in silico supports thatthe iteron-bound RepA monomers (mRepA) are directlyinvolved in the proteinprotein contacts relevant forhandcuffing.

Results

Iteron-bound RepA builds looped and handcuffedplasmid assemblies in vitro

To visualize by TEM any large complex formed by RepA atthe pPS10 operator and the iterons, we studied each DNAsequence separately using purified RepA-WT protein aswell as two pUC18 derivatives each carrying one type ofrepeats (Daz-Lpez et al., 2003). The proteinDNA com-plexes were reconstituted with linearized plasmids.For the operator, DNA showed a defined globular protein

particle at a position compatible with being located atthe cloned inverted repeat (Fig. 2A, left). A graph showingthe distribution along the plasmid of the experimentallydetermined binding sites (Fig. 2A, right) locates asecond site in the pUC18 vector, outside the clonedpPS10 sequences. Its position roughly corresponds to a5-GGACAG sequence, coincident with half of the opera-tor inverted repeat and with the 3-end of an iteron directrepeat (Giraldo et al., 1998). The fact that this subsidiarysite is barely populated reflects the intrinsic low affinity ofRepA dimers (dRepA), the most abundant molecularspecies in solution, for a single 5-GGACAG repeat (Daz-Lpez et al., 2006). In contrast, binding of RepA to thecloned four iteron repeats (Fig. 2B, left), where it stablybinds as monomer (mRepA) (Giraldo et al., 1998), resultsin three main kinds of assemblies: linear, as in thecomplex with the operator sequence; handcuffed, twoplasmid molecules stuck together; or lariats, lasso typecomplexes made by folding back RepA-bound iterons onthe distal vector 5-GGACAG sequence.As expected from

the low DNA concentrations in which they are assembled,the most abundant complexes are intramolecular loops(Fig. 2B, left).

An issue relevant for the structure of handcuffing com-plexes is the relative orientation of the iteron arrays thatare brought together through RepARepA interactions.For the P1 replicon, it was inferred, from the effect onplasmid copy-number of an extra iteron repeat cloned inits two possible orientations, that both iteron arrays werecoupled in parallel orientation (Das and Chattoraj, 2004).Our TEM micrographs of RepA-handcuffed pPS10iterons show complexes with a pseudo-twofold symmetry

axis along the coupled DNA fibres (Fig. 2B, left, centralpanel). The two shortest of the four DNA tails that pro-trude away from the RepA particle are always locatedtogether, side by side and opposite to the two longestones. For the R6K plasmid, similar complexes wereassigned to a parallel orientation of the coupled iteronsarrays (Urh et al., 1998). To confirm that the DNA tailsappearing to have similar lengths were indeed identical,we performed careful measurements of their lengths inseveral handcuffed complexes. We thus ruled out thateither of the tails in one of the linear plasmid moleculeswas wrapped around a RepA core: the iterons would bethen in antiparallel orientation, but having the appear-

ance shown in Fig. 2B. Thus, as in P1, the formation oflariats (Fig. 2B, left, bottom panel) can be also attributedto parallel interaction between two oppositely orientedDNA sequences. When we reconstructed both hand-cuffed and lariat assemblies with a plasmid linearized inan alternative single restriction site, which leaves theiteron array centred and the 5-GGACAG repeat close toan end, we obtained similar results as expected (notshown).

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A first indication of a role for the N-terminal WH1 domainin handcuffing comes from RepA-2L2A, a double mutantthat has the stability of such a domain severely reduced(Daz-Lpez et al., 2003), because it fails to establisheither lariat or handcuffed complexes (Fig. 2C). The sizeof the RepA-2L2A particle at the iterons is larger than thatof the WT protein (Fig. 2B, top), as expected from theenhanced tendency of this mutant towards aggregation(Daz-Lpez et al., 2003).

A proteomic dissection of RepA-DNA

supramolecular assemblies

It has been implied that distinct molecular species ofRepA participate in the complexes established at opera-tor and iteron DNA, and that the protein uses differentcontacting interfaces for each context (Giraldo et al.,2003; Giraldo and Fernndez-Tresguerres, 2004). Theseinclude residues involved in dimerization, in contacts

Fig. 2. Macromolecular assembliesestablished by RepA at the operator anditeron sequences visualized by TEM.A. Left: RepAoperator complexes in twolinearized plasmid molecules. Right: Plot withthe statistical distribution of protein complexesalong the DNA molecule, highlighting thepositions of the operator (symmetric arrows)and a vector sequence identical to anoperator/iteron half (arrowhead).

B. Left: Views of the three distinct complexesmade by RepA in a plasmid carrying the fouriteron repeats. Right: The distribution of RepAparticles along the linear plasmid, as in (A).The iteron array (arrows in tandem) and thehomologous vector sequence (arrowhead) aremarked. The relative abundance of each kindof complex is indicated. Arrows: Bound RepAparticles.C. The metastable RepA-2L2A (WH1) mutant(Daz-Lpez et al., 2003) binds to plasmidDNA at the cloned four iterons sequence, butis unable to couple (or loop) DNA molecules.Magnification bars: 100 nm.




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between monomers bound at contiguous iterons or inhandcuffing. To test this hypothesis by mapping proteinareas in close spatial proximity, we cross-linked thedifferent RepADNA complexes with disuccinimidylglutarate (DSG), a bifunctional reagent for free amino

groups. The covalent protein assemblies were thenresolved by SDS-PAGE (Fig. 3A). Six protein bandswere analysed, including RepA monomers (#1) anddimers, the latter either free in solution (#2) or incomplex with operator DNA (#3). In addition, three

Fig. 3. Proteomic mapping of proximalprotein surface residues in the distinctRepARepA assemblies.A. RepA and RepADNA complexes werecross-linked with DSG and then resolved bySDS-PAGE. Bands corresponding tomonomeric (mRepA; #1), dimeric (dRepA;#23) and handcuffed proteins (hc13; #46)were excised, treated with trypsin and theresulting peptides were eluted.

B. MALDI-TOF mass spectra of the peptidesgenerated in A from gel bands #16. Theexperimental masses of the peaks and theirCorrespondence tryptic peptides identified inRepA are indicated. Blue: Peaks arising fromDSG-cross-linked peptides located in theWH1 domain but only in the dimeric RepAspecies (dRepA); red: peaks exclusive forRepA monomers (mRepA). Green:cross-linked peptides that came from bothWH1 and WH2.




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bands with lower electrophoretic mobility (#46) thatexclusively appeared when RepA was incubated withthe array of four iterons were analysed. According totheir electrophoretic mobility, these bands correspondto assemblies of two (#4 and #5) and four (#6) RepAmonomers. Protein bands were analysed by proteolysisin-gel, followed by MALDI-TOF mass spectrometry ofthe resulting peptides (Fig. 3B). Spectral peaks derivedfrom the straight digestion of RepA were initially identi-fied and those whose identities were still missing wereassigned to defined cross-linked peptide pairs (fordetails, see Experimental procedures).

A first glimpse of the peaks identified as cross-linkedpeptides makes evident that some exclusively appearassociated to the dimeric state of RepA (Fig. 3B, free andoperator-bound protein, bands #2 and #3, in blue). Othersare only found related to complexes established by RepAat the iteron arrays (Fig. 3B, bands #46, in red), wherethe protein is known to bind stably as monomers (Giraldoet al., 1998; Daz-Lpez et al., 2003). These peptides are

all located in the WH1 domain, as expected from its role indimerization (Giraldo et al., 1998; 2003) and in handcuff-ing (Fig. 2C). In addition, several peaks generated byWH1-WH2 cross-linking were found (green). Thesepeptides could be due to intrasubunit (in dimers) and/orintermolecular bridges (between monomers assembledhead-to-tail). No cross-linked peptides arose from WH2WH2 interactions.

Screening for RepA mutants impairing plasmid

incompatibility in vivo

To search for RepA mutants with handcuffing abilitiesreduced in vivo (Fig. 4A), we took advantage of the inti-mate relationship between handcuffing and the incompat-ibility between plasmid vectors carrying identical copiesof a given iteron array. For this purpose, we mutateda pPS10 (kanamycin-resistant, Knr) derivative in vitrowith hydroxylamine (HA) and then transformed it intoPseudomonas aeruginosa cells carrying a RK2 mini-plasmid (tetracycline-resistant, Tcr). Because the fourpPS10 iterons were cloned into the latter, the two plas-mids were incompatible in the absence of selectivepressure with both antibiotics. The initial plating of thetransformants on media including both markers allowed

counter-selecting mutations deleterious for pPS10, eitherin repA, oriV or the resistance determinant. The persis-tence of the resistance to Tc, and thus the presence of theRK2 derivative, was checked, after a number of serialpasses in the absence of this antibiotic, by a final platingon Kn and Tc. Clones with a Knr but Tcs phenotype shouldhave lost the RK2 plasmid, displaced by handcuffingexerted in trans by RepA on the iterons cloned in thevector. Cells that grew in the presence of both antibiotics

retained pPS10 and RK2 vectors, owing to the inability ofa mutant RepA protein to handcuff their iterons. We puri-fied plasmids and sequenced the pPS10 replicon from 24clones that exhibited the latter phenotype. We identifiedfour different repA mutants, all showing the C,T or G,Atransitions characteristic of the mutagen used, encodingfor: A31V (54% of clones), A31T (21%), T47I (12.5%)and P113S (12.5%). Their stabilities and relative copynumbers were then determined (Fig. 4B). We found thatall of them were as stable as the WT, and showed copynumbers between two and three times that for the WT, asexpected for mutations not directly affecting replicationinitiation but its control.

In addition, K87E, a mutant affecting a residue foundby proteomics to be a marker for mRepA-mediated hand-cuffing (Fig. 3B), was constructed by site-directedmutagenesis. Its phenotype, in terms of stability, copynumber and ability to coexist with the RK2 derivative, wasindistinguishable from that reported above for the repAmutants induced by HA (Fig. 4B). These results are con-

sistent with the view that RepA monomers are involved inhandcuffing.

RepA mutants lead to a reduction in the number of

looped and handcuffed assemblies

To assess whether any of the mutations that affected copynumber control has altered the overall structure of RepA,we overexpressed and purified the RepA mutants (Fig.S1A). We then determined their association state bygel filtration analysis (Fig. S1B) and estimated theiraverage secondary structure and thermal stability by

means of circular dichroism spectroscopy (Fig. S1C andD respectively). Our results indicate that there is no majorstructural difference between them and RepA-WT. Inaddition, DNA binding of the mutants, both to the repAoperator and to the array of four iterons, was virtually thesame than for RepA-WT (Fig. S2). Therefore, the muta-tions must be acting at a level other that altering RepAstructure or its binding to DNA.

We also explored by TEM the possibility that mutationswere hampering the assembly of RepA into the regulatorycomplexes shown in Fig. 2. The distribution of the par-ticles observed among the three main classes of assem-blies (linear, lariat and handcuffed) (Fig. 5) indicates that

most of the mutations drastically increase the frequencyof linear complexes at the cost of a reduction in thenumbers of both lariats and handcuffs. However, somemutations exhibit a differential effect: A31T andA31V leadto a larger decrease in the frequency of handcuffed com-plexes than of lariats (these even go up for A31T),whereas T47I, K87E and P113S have a drastic effect onboth types of complexes, being most extreme for the lasttwo mutants.




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Fig. 4. Search for RepA mutants defective incontrol of pPS10 replication.A. Schematic outline of the procedure forselecting compatible replicons. Grey: lostplasmid. Asterisk: mutant repA.B. Histogram showing, after 120 generationsof growth without selective pressure, the copynumber, compatibility and stability phenotypesof pPSEC2 variants encoding for the indicated(vertical axis) RepA mutations. Numbers in

the horizontal axis were normalized relative tothe value for a plasmid carrying RepA-WT.




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A 3D model for handcuffed plasmid origin assemblies

by pairing iteron-bound RepA monomers

The Lys residues identified to cross-link and theHA-mutations reducing handcuffing were located in thecrystal structure of the dimeric WH1 domain (Giraldoet al., 2003) (Fig. 6A) and in a series of models formonomers brought in interaction that were built by means

of automated docking in silico (see Experimentalprocedures). Figure 6B shows the model with the best fitto the restrictions in distances coming from the cross-linking data (Table 1). As observed by TEM (see above),the selected model corresponds to a parallel arrangementof iteron-bound monomers held together by interactionsthrough their WH1 domains.

In dRepA, the residues identified to cross-link aremostly found in the large loop located C-terminal to a2(K38 and K42) (Fig. 6A, blue spheres), the most flexibleregion in the crystal structure of WH1 dimers (Giraldoet al., 2003). Our results show that the key residues dis-criminating between dRepA and mRepA in handcuffing

are K85 and K87 (red spheres). In distinct subunits of adimer, these residues are too far apart to be cross-linked.Furthermore, there is no sign for any mass attributable toan intrasubunit cyclic adduct having their e-NH2 groupslinked together. On the contrary, in the modelled assemblyof monomers (Fig. 6B), K85 and K87 are arranged atthe appropriated distances to get all the observed cross-linked adducts, either between handcuffed monomers(interchain) or between a monomer and the nearest

neighbour to its handcuffed partner (zigzag) (Table 1).When the HA-mutated residues (cyan spheres) weremapped in the structure of a RepA-WH1 dimer (Fig. 6A)and in the model for two handcuffed monomers (Fig. 6B),it was found that they are closer to the contacting interfacebetween iteron-bound mRepA molecules than to thedimerization interface in dRepA. This suggests that themutated residues impair handcuffing by altering inter-monomer contacts.

Overall, mapping both the HA-mutated residues andthose found to cross-link in a proteomic analysis of theprotein-DNA assemblies associated with handcuffing iscompatible with a model in which RepA monomers aredirectly in contact through an interface distinct from thatknown to be used in dimerization.

Discussion

Using an approach that combines electron microscopy(Fig. 2) with protein cross-linking, proteolysis and mass

spectrometry (Fig. 3), we characterized the regulatorycomplexes (intramolecular loops and intermolecular

Fig. 5. Histogram summarizing the distribution, among the three

different classes of assemblies visualized by TEM (Fig. 2B) of thecomplexes between RepA variants and the four iterons fragment.The total number of particles counted for each class is displayedbelow the x-axis.

Fig. 6. RepA residues relevant for handcuffing mapped in 3D.A. The crystal structure of a dimer of RepA-WH1 (Giraldo et al.,2003). Subunits: yellow and magenta. CPK spheres: theside-chains of residues found to cross-link in RepA dimers (blue) ormonomers (red). Cyan: residues found to be mutated in plasmidsthat became compatible after random mutagenesis (see Fig. 4).B. Model for two handcuffed iteron (orange)-bound RepAmonomers (WH1s in yellow and magenta; WH2s in blue andgreen). Arrows highlight the parallel orientation of the iterons.




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handcuffs) assembled by the replication protein RepAupon binding to the pPS10 plasmid iteron repeats. Weisolated and characterized, in vivo and in vitro, RepAmutants (Fig. 4) with an impaired ability to handcuff orloop iteron arrays (Fig. 5). These RepA variants remaindimeric in solution (Fig. S1B), but the mutated residuesmap at a new interface for the interaction between RepAmonomers that we identified by proteomics (Fig. 3) andthen modelled by protein docking in silico (Fig. 6B).

This work provides the first indication for the differentnature of the DNA-RepARepA-DNA complexes involved

in intramolecular looping and intermolecular handcuffing,so far thought to be equivalent (Zzaman and Bastia, 2005;Paulsson and Chattoraj, 2006). The potential of the RepAmutants to loop distant iterons in cis is less affected thantheir ability to handcuff iteron arrays segregated in trans(Fig. 5). Although this could be in part reminiscent of theirdistinct frequencies in the RepA-WT control, perhapsreflecting the higher probability for any intramolecularcomplex, it could be also related to the fact that the lattertype of assemblies is built on the interaction betweenRepA monomers (Figs 3 and 6). We propose, however,that dimers would be the Rep protein species acting in theassembly of intramolecular loops.

It has recently been reported that the A31V mutationdrives the DNA-promoted assembly of RepA-WH1 intospheroids and fibres with the cross-b structure character-istic of protein amyloids (Giraldo, 2007). This mutationalso contributes to a more efficient interaction of RepAwith host factors, thus broadening pPS10 host range(Fernndez-Tresguerres et al., 1995; Maestro et al.,2003). We here report that this dimeric RepA mutant,having a potential for amyloid aggregation, is unable to

handcuff, although it exhibits little reduction in DNAlooping, which even increases in the case of the relatedmutant A31T (Fig. 5). Because the RepA-A31T mutationresults in the highest plasmid copy number (Fig. 4B), itfollows that handcuffing assemblies are indeed the majorplayers in pPS10 replication control.

As only the 1.8 kbp basic replicon has been sequencedin pPS10 so far (Nieto et al., 1992), the presence ofextra, yet unidentified, iteron (inc) repeats in the remain-ing 8.2 kbp of the plasmid is still possible. These repeatscould be involved in replication control through looping in

cis mediated by RepA dimers, analogous to the complexdescribed between oriV iterons and the half iteron repeatfound in the cloning vector used in the TEM experiments(Fig. 2). However, the mini-plasmid used in our experi-ments in vivo, in which just the minimal replicon remainsfrom pPS10, seems able to keep its copy number stable(Fig. 4), based on repA self-repression and iteron titration/handcuffing. The absence of RepA-bridged DNA lariatswhen the protein molecules bind to the operator sequence(Fig. 2A) reflects that a RepA dimer engages its two WH2domains in a complex with the two halves of the operatorinverted repeat (Giraldo et al., 1998; Daz-Lpez et al.,2006), and no DNA binding domain is thus available to

interact at a distance. However, the inability of the opera-tor DNA to induce the conformational activation of RepA(Daz-Lpez et al., 2003) could also point to the require-ment of some sort of structural change in the protein inorder to exert handcuffing, such as that coupled to RepAmonomerization (Giraldo et al., 2003).

In Fig. 7 the model for two iterons coupled in parallelorientation by DNA-bound RepA monomers interactingthrough their WH1 domains (Fig. 6B) has been extended

Table 1. Shortest distances measured between the e-amino (NZ) groups of the Lys residues that were found cross-linked (Fig. 3B) in the modelsfor the RepA-WH1 domain dimer (Fig. 6A) and handcuffed RepA monomers (Figs 6B and 7).

Residuesa Band #b NZ-NZ () Typec

dRepA K85 K85 55.1 InterchainK85 K87 48.4 InterchainK87 K87 43.4 InterchainK38 K38 2, 3 17.9d InterchainK38 K42 2, 3 12.8d Interchain

K38 K99 2 18.7d Interchain2(mRepA + iter)4 K85 K85 6 19.2/ 9.3 Interchain/zigzag

K85 K87 4, 6 12.6/14.0 Interchain/zigzagK87 K87 5, 6 7.3 ZigzagK38 K38 37.8 InterchainK38 K42 28.7 InterchainK38 K99 19.7 Intrachain

a. Italics, dimeric assemblies; bold, assemblies involving RepA monomers.b. The identities of the analysed gel bands (#26; Fig. 3A). Distances within a range compatible with the experimental cross-linking results(719 ) are highlighted in bold.c. Interchain, two distinct molecules, either the subunits in dimers or the monomers in a handcuffed complex; intrachain, between residues in thesame molecule; zigzag, between monomers bound to iterons in opposite DNA fibres, but staggered by one step.d. Alternative side chain rotamers were tested in a high B-factor (6695 2) loop; thus these must be considered average distances.




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to the natural array of four contiguous iterons found at

the pPS10 replication origin. This arrangement results in aright-handed twisted protein-DNA superhelix. As it isevident from the dimensions of the protein particles in theelectron micrographs showing handcuffed supramolecu-lar assemblies (Fig. 2), handcuffing complexes couldcomprise many more RepA molecules bound than theeight monomers modelled in Fig. 7. Indeed, such a modelhas plenty of room for additional RepA molecules at thegrooves of the superhelix. Although it should be notedthat the cross-linked peptides acting as a signature forRepA dimers are absent in the handcuffed protein bands(Fig. 3B), it could be still possible that protein dimerswould subsequently contribute to negative control of

plasmid replication together with the primary layer ofmonomers. The latter scenario could be relevant in thesupercoiled state in which plasmids are found in vivo, butsuch hypothesis needs to be addressed experimentally.

Plasmid Rep initiators are among the most versatileDNA binding proteins known, because their ability totackle multiple functions, such as transcriptional repres-sion and replication initiation/inhibition. Rep proteinsswitch between roles by undergoing substantial structural

changes, including dimer dissociation, interdomainmotion and conformational changes, that enable Repmonomers to assembly into specialized nucleoproteincomplexes, such as those shown in this work to handcufftwo iteron DNA arrays.

Experimental procedures

RepA and DNA purification

Both WT and mutant His6-tagged RepA proteins were over-expressed in E. coli using the pRGrectac-NHis vector. Theoverexpressed proteins were then purified by Ni2+-affinitychromatography (Agarose Beads Technologies) and theirconcentration was determined, as previously described(Giraldo et al., 1998; Giraldo, 2007). DNAs for electrophoreticmobility-shift assays (EMSA) and protein cross-linking weregenerated by PCR, using as templates pUC18 derivatives(0.1 mg) carrying either a 39 bp sequence spanning the repAoperator or 88 bp, comprising the four pPS10-oriV iterons,both cloned into SmaI (pUC-oriV0 and pUC-oriV4 plasmidsrespectively; Daz-Lpez et al., 2003). The f17 and r19 uni-versal primers (50 pmol each) were used for 40 cycles ofamplification using the gel-adsorbed Taq DNApol (BioTools).Amplified dsDNAs were purified by means of phenol-chloroform-isoamylic alcohol extraction followed by ethanolprecipitation.

Transmission electron microscopy

pUC-oriV0 and pUC-oriV4 were digested with NdeI (orAlwNI), to give linear plasmids. Their ends were blunt-endedwith Klenow and purified as described above. Each RepADNA complex of 40 ng ml-1, made at equimolar concentra-tions, was incubated for 15 min at 4C in 10 ml of reaction

buffer containing 0.1 M KCl, 25 mM Tris-HCl pH 7.5, 10 mMMg-acetate, 4% sucrose, 1 mM ATP and 1 mM DTE. Thecomplexes were fixed with 0.2% glutaraldehyde for 15 min at4C and then diluted to 40 ml with 10 mM triethanolamine-HClpH 7.5 and 10 mM MgCl2. Samples were then adsorbed tomica and positively stained with 2% uranyl acetate. Rotaryshadowing was carried out with platinum and then grids werecovered with a carbon film as described (Spiess and Lurz,1988). Micrographs were taken using a Philips electronmicroscope CM100 (FEI, Hillsboro, Oregon, USA), operatingat 100 kV, and a Fastcan CCD camera (Tiez Video and ImageProcessing Systems GmbH, Gauting, Germany). To deter-mine the DNA contour length and the position of RepAbinding sites, distances were measured on 35 mm films with

a digitizer (LM4, Brhl, Nuremberg, Germany).

Protein cross-linking and proteomic analysis of

RepADNA complexes

RepAoperator and RepA-4 iteron complexes wereassembled in 40 ml reactions containing 5 mg (4.5 mM) ofHis6-RepA, various amounts of the specific DNAs (at bindingsites : mRepA molar ratios ranging from 0.5 to 2.5) in 0.15 MNa2SO4, 0.02 M Hepes-NaOH pH 7.0, 10% (v/v) glycerol for

Fig. 7. Right-handed superhelical protein-DNA assembly resultingafter modelling two DNA fibres handcuffed, in parallel orientation,by interactions established between two arrays of iteron-boundRepA monomers (Fig. 1B, centre). Colours are as in Fig. 6.




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15 min on ice. Full RepA binding to DNA was checked inparallel by means of EMSA (5% polyacrylamide-0.25 TBEgels, EtBr staining; not shown). Then 1 ml of 4 mM DSG(Pierce) in dimethylsulphoxide was added and further incu-bated for 45 min at room temperature. Reactions werestopped with 1 ml of 1 M Tris-HCl pH 8.0 and samples wereimmediately analysed by SDS-PAGE (12.5% polyacrylamidegels) with silver staining (SilverQuest kit, Invitrogene). Bandsto be analysed were excised from the gel, dehydratedwith acetonitrile and then left soaking in 100 ml of 25 mMNH4HCO3 pH 8.9 in ice, supplied with 0.02 unit of bovinetrypsin. Proteolysis in situ was achieved after replacing theexcess of trypsin with bicarbonate buffer by overnight diges-tion at 37C. Peptides were then eluted from the gel slices byserial extractions with 50% acetonitrile/0.5% TFA and pureacetonitrile. Mass spectra of the peptides found in theextracted fractions were acquired by mixing 0.5 ml of eachsample with a saturated matrix solution of a-cyano-4-hydroxycynnamic acid in acetonitrile:0.1% TFA (1:2).Samples were then analysed in a Biflex-III MALDI-TOF massspectrometer (Bruker-Franzer Analytik, Bremen, Germany).Each spectrum is the average of 100 accumulated lasershots. Calibrations were made with a mixture of angiotensin(m/z = 1046.54 Da) and ACTH (m/z = 2465.20 Da). Peptidemasses were then compared with those predicted (http://www.expasy.ch/tools/peptide-mass.html) from the trypticdigestion of His6-RepA, allowing for several missedcleavages. Orphan peaks that did not fit with RepA (excludingprotein-DSG-Tris adducts, self-digestion products of trypsinor keratins) were classified as possible cross-linked peptides.These peptides were identified by comparing their experi-mental masses with the combined masses of any possiblepair of tryptic RepA peptides including at least an internalundigested Lys residue (or the protein N-terminus) plus the1,5-pentanedione link (98.1 Da).

Hydroxylamine and site-directed mutagenesis of repARandom mutagenesis with HA was performed as described(Fernndez-Tresguerres et al., 1995; Maestro et al., 2003). Insummary, 20 ml (4 mg) of the pPS10 Knr derivative pPSEC2(Daz-Lpez et al., 2003) were mixed with 100 ml of 0.1 MNa-phosphate pH 6.0, 1 mM EDTA and 80 ml of 1 M HA in thesame buffer. The mixture was incubated at 75C for 30 minand then dialysed at 4C against 2 500 ml of TE buffer (for2 and 12 h respectively). DNA was electroporated (accordingto the specifications of the manufacturer, Bio-Rad) intoP. aeruginosa PAO1024 cells containing pCM51-oriV4, amini-RK2 Tcr derivative (Marx and Lidstrom, 2001). pCM51-oriV4 had the pUC-oriv4-derived EcoRI-HindIII fragment,whose ends had been filled with Klenow, cloned into its AfeIsite. Cells were immediately suspended in SOC medium andincubated at 37C for 1 h. Transformants were selected onLB agar plates, supplemented with Tc (10 mg ml-1) and Kn(75 mg ml-1) and tested for RepA-mediated compatibility (seebelow). To obtain the RepA mutant K87E, site-directedmutagenesis was performed using of the PCR-based Quick-Change Kit (Stratagene) and pPSEC2 as template. Theprimers for this reaction were (mutated Glu codon under-lined): 5-GCAGGTACGTCAAAGGCGAAGTCGTTGAACGCATGCG, plus its complementary strand. In all cases, muta-

tions were verified by DNA automated sequencing (AppliedBiosystems ABI3730), using primers annealing 5 or 3 to therepA gene.

Plasmid incompatibility, stability and copy

number determination

After the HA mutagenesis procedure, transformants that grew

in LB + Tc + Kn were replica-plated 34 times (approximately90120 generations) into agar plates and selected for thepPS10 replicon (Kn) but not for the oriV4-carrying RK2 (Tcomitted). Colonies that were able to grow in the presence ofboth antibiotics were selected for DNA sequencing as poten-tial repA mutants impaired in handcuffing (see above). Thestability of each pPSEC2-repA mutant was checked by patch-ing, on LB and LB + Kn agar, 100 independent colonies froma single transformant previously grown in LB agar, thus deter-mining the number of antibiotic-sensitive colonies. This pro-tocol was carried out for up to four passes (approximately 30,60, 90 and 120 generations). The effect of the mutationsin plasmid copy number was checked by preparing totalcell lysates obtained from exponentially growing cultures

of P. aeruginosa PAO1024 (Nieto et al., 1992) containingpPSEC2 (repA-WT), or the same plasmid carrying the repAmutants described in this work. Samples were electrophore-sed in 0.8% agarose-TAE gels for 3.5 h at 33 V. The relativeamounts of plasmid and chromosomal DNA in the EtBrstained bands were estimated by densitometric analysis,using the Quantity One software (Bio-Rad Gel Doc 2000).

Modelling RepARepA assemblies by protein

docking in silico

RepA monomers were modelled on the structure of thehomologous monomeric initiator RepE54 (PDB code 1REP;

Komori et al., 1999) by means of the Swiss-Model server(http://swissmodel.expasy.org; Guex and Peitsch, 1997) asdescribed (Giraldo and Fernndez-Tresguerres, 2004). Twosuch mRepAs were brought to interaction by the automatedprotein docking procedure implemented at the ClusProserver (http://nrc.bu.edu/cluster/) by initially selecting thesolutions with the highest score among those generated bythe docking programs provided (DOT and ZDOCK, 15 solutionseach) (Comeau et al., 2004). An iteron was then manuallyfitted to the a4 and a4 binding helices in each dockedmRepA, maximizing its superposition with DNA in theRepE54 crystal structure (Komori et al., 1999). The 30 pre-selected models were then filtered according to the followingcriteria: (i) solutions showing evident steric problems werediscarded (e.g. those docking through DNA binding surfaces,with no room for DNA, or through the WH2 domains). (ii) Thedistances between the cross-linked residues should havethe best possible fit to the proteomic data (Table 1). (iii) Themutated residues had to be close to the mRepAmRepAinterface. At this stage, just two models were still kept. (iv)The orientation of proteins and DNA fibres in the complexshould be parallel. This procedure left DOT model 3 (Fig. 6B)as our working model for two handcuffed RepA monomers.This model is only tentative, as the docking procedure doesnot currently allow for any flexibility in the structure of the


http://www.expasy.ch/tools/peptide-mass.htmlhttp://www.expasy.ch/tools/peptide-mass.htmlhttp://swissmodel.expasy.org/http://nrc.bu.edu/clusterhttp://nrc.bu.edu/clusterhttp://swissmodel.expasy.org/http://www.expasy.ch/tools/peptide-mass.htmlhttp://www.expasy.ch/tools/peptide-mass.html


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interacting proteins. The model for the two handcuffed arraysof iterons bound by four mRepA molecules each (Fig. 7) wasbuilt using Swiss Pdb-Viewer (Guex and Peitsch, 1997) byadding, head-to-tail, three additional copies of the selectedClusPro-DOT model including an iteron bound at eachmRepA, in such a way that iterons arranged into two continu-ous DNA fibers. Models were rendered with PyMOL (http://pymol.sourceforge.net/).

Acknowledgements

We thank Dr M.E. Lidstrom for the gift of the mini-RK2 vectorpCM51. We are also grateful to Professor R. Daz-Orejas andDr Angie Hofmann for the critical reading of the manuscript.This work has been financed by grants of Spanish MEC(BMC2003-00088 and BFU2006-00494). The temporal staysof F.G.-R. and T.D.-L. in the laboratory of R.L. have beensupported, respectively, by a CSIC-MPI exchange and by theCSIC Program for International Cooperation 143A.

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