structural biology of plasmid partition: uncovering the ......dna segregation or partition is an...

Biochem. J. (2008) 412, 1–18 (Printed in Great Britain) doi:10.1042/BJ20080359 1

REVIEW ARTICLEStructural biology of plasmid partition: uncovering the molecularmechanisms of DNA segregationMaria A. SCHUMACHER1

Department of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Cancer Center, Unit 1000, Houston, TX 77030, U.S.A.

DNA segregation or partition is an essential process that ensuresstable genome transmission. In prokaryotes, partition is bestunderstood for plasmids, which serve as tractable model systemsto study the mechanistic underpinnings of DNA segregation ata detailed atomic level owing to their simplicity. Specifically,plasmid partition requires only three elements: a centromere-like DNA site and two proteins: a motor protein, generally anATPase, and a centromere-binding protein. In the first step of thepartition process, multiple centromere-binding proteins bind co-operatively to the centromere, which typically consists of severaltandem repeats, to form a higher-order nucleoprotein complexcalled the partition complex. The partition complex recruits theATPase to form the segrosome and somehow activates the ATPasefor DNA separation. Two major families of plasmid par systemshave been delineated based on whether they utilize ATPaseproteins with deviant Walker-type motifs or actin-like folds. Incontrast, the centromere-binding proteins show little sequencehomology even within a given family. Recent structural studies,however, have revealed that these centromere-binding proteinsappear to belong to one of two major structural groups: thosethat employ helix–turn–helix DNA-binding motifs or those with

ribbon–helix–helix DNA-binding domains. The first structure ofa higher-order partition complex was recently revealed by thestructure of pSK41 centromere-binding protein, ParR, bound to itscentromere site. This structure showed that multiple ParR ribbon–helix–helix motifs bind symmetrically to the tandem centromererepeats to form a large superhelical structure with dimensionssuitable for capture of the filaments formed by the actin-like ATPases. Surprisingly, recent data indicate that the deviantWalker ATPase proteins also form polymer-like structures, sug-gesting that, although the par families harbour what initiallyappeared to be structurally and functionally divergent proteins,they actually utilize similar mechanisms of DNA segregation.Thus, in the present review, the known Par protein and Par–protein complex structures are discussed with regard to theirfunctions in DNA segregation in an attempt to begin to define,at a detailed atomic level, the molecular mechanisms involved inplasmid segregation.

Key words: deviant Walker box motif, DNA segregation,insertional polymerization model, partition, partition complex,plasmid.

INTRODUCTION

DNA partition or segregation, is the process whereby the geneticmaterial is accurately moved and positioned to daughter cellsduring cell division. The partition of newly replicated eukaryoticchromosomes is carried out by the formation of microtubule-based spindles, which pull chromosomes to opposite cell poles[1]. In contrast, the molecular machinery that mediates thesegregation of prokaryotic DNA has been less clear. Much of ourunderstanding of prokaryotic partition has resulted from cellular,genetic and biochemical analyses on the segregation of low-copy-number plasmids. Such plasmids play a significant role in thespread of multidrug resistance and also play a central role in DNAmanipulation technology. Thus understanding the mechanismbehind their maintenance has important consequences. Whereashigh-copy-number plasmids primarily rely on passive diffusionfor plasmid maintenance, low-copy-number plasmids utilize so-called partition (par) systems, which are carried on the plasmidDNA [2–13]. The majority of par operons or cassettes containtwo genes: one encoding a motor protein, which is typicallyan ATPase, and the second encoding a centromere-bindingprotein [2–11]. In addition, the centromere-like site bound bythe centromere-binding protein is located near the par cassette.These three components are all that is required to direct the

partition reaction (Figure 1A). However, the amounts of each ofthe Par proteins are carefully regulated, as deficiency or excessof either protein can completely or seriously impede the partitionprocess [14–17]. Fluorescence microscopy studies have provideda visual picture of the highly dynamic partition reaction. Forthe most common type of par system, immunofluorescence andFISH (fluorescent in situ hybridization) studies have shown thatnewborn cells usually have one central plasmid focus, whereaslarger older cells have two foci located at the one-fourth andthree-fourth positions of the cell, indicative of plasmid segregation[18–23]. Studies using GFP (green fluorescent protein)-taggedplasmids gave visualized plasmid movement in live cells and haverevealed that plasmid movement and separation are relativelyrapid events in the entire cell cycle. The challenge has been torelate these visual observations with the molecular steps involvedin plasmid partition.

A general understanding of the steps involved in DNA partitionhas been obtained from biochemical, cellular and genetic studies.Specifically, the first step involves binding of the centromere-like partition site by the centromere-binding protein, ultimatelyleading to the creation of a large nucleoprotein complex [3–11](Figure 1A). Next, interactions between the centromere-bindingproteins and the DNA lead to pairing between partition complexeson two different plasmids. Once the plasmids are paired, the

Abbreviations used: EM, electron microscopy; FP, fluorescence polarization; HTH, helix–turn–helix; IHF, integration host factor; p[NH]ppG, guanosine5′-[β,γ-imido]triphosphate; RHH, ribbon–helix–helix; SH3, Src homology 3.

1 email [email protected]

c© The Authors Journal compilation c© 2008 Biochemical Society

2 M. A. Schumacher

Figure 1 Genetic organization of par operons

(A) Schematic diagram of the general steps of plasmid partition. Plasmids are represented as large circles, and the partition complexes formed by centromere-binding proteins binding to thecentromere are shown as small circles. The first step is partition complex formation. Subsequently, plasmid pairing (interacting ovals) occurs. Once the plasmids are paired, the motor protein isrecruited to the partition complex and drives plasmid separation. (B) Genetic organizations for the two main families, type I and II, of par loci. Shown are representatives from each class (type Ia, Fand P1; type Ib, pTP228; double loci, pB171; and type II, R1). Orange arrows represent genes that encode motor proteins (ATPases) and blue arrows represent genes encoding centromere-bindingproteins. Centromere sites are indicated by black bars and are labelled (e.g. parS, sopC, parC1, parC2 and parC). Arcs represent DNA-binding properties of the indicated gene products, continuousarcs show regulation of promoter activity by the indicated gene products and broken arcs represent formation of partition complexes.

partition complexes are recognized by the motor protein leadingto the assembly of the segrosome, which then actively mediatesthe separation of the plasmids (Figure 1A). The simplicity of theplasmid par systems marks them as extremely attractive modelsystems to address the fundamental biological question of howDNA is segregated to daughter cells at the atomic level. Asdescribed in the present review, structural work carried out inthe last 5 years has started to elucidate the detailed molecularmechanisms utilized by these proteins in each step of the DNAsegregation process.

PLASMID PARTITION CASSETTES AND FAMILIES

The first identified plasmid partition systems and their associatedgenetic loci are those of the Escherichia coli P1 and F plasmidpar systems. These discoveries were made over 25 years ago bythe Hiraga and Austin groups [11,24,25]. Since then, multiple parcassettes have been found experimentally and by bioinformaticsapproaches in plasmids from very diverse bacteria [7]. Most ofthese cassettes have very similar genetic organizations wherebythey consist of three elements: a cis-acting centromere-like siteand two trans-acting proteins: a centromere-binding protein anda motor or force-generating protein (Figure 1B). Although theforce-generating proteins were all originally found to be ATPases,recent work shows that tubulin-like GTPases and, possibly, coiled-coil proteins can also function as the force-generating proteinto drive partition. To reflect this, these proteins are referred toas motor proteins. The upstream gene in the operon encodesthe motor protein, whereas the downstream gene encodes thecentromere-binding protein (Figure 1B). The centromere-like siteitself is typically located either upstream or downstream of thepar operon [3–11]. An important property of par cassettes is theirability to stabilize heterologous replicons, which means that theycan act independently of the replication system and still mediateplasmid separation, ensuring plasmid maintenance.

An analysis of all the par systems that had been identified todate by Gerdes et al. in 2000 [7] led to their classification intotwo main types. This categorization was based on the kind ofmotor protein that is present, as the centromere-binding proteinsexhibit significant sequence diversity. Accordingly, the par motorproteins can readily be broken into two main groups. In thesetwo groups, the motor protein belongs to one of two familiesof ATPases: one with a deviant Walker box motif (also calledP-loop) and the second containing an actin-like fold. In the presentreview, this categorization of par systems as suggested by Gerdeset al. [7] has been used because it provides a useful frameworkfor describing and comparing Par protein structure. More recently,additional putative par systems that are unrelated to the two majorpar families have been identified. However, structures of theseproteins have yet to be determined. We shall begin by definingand describing the two par major families as well as two morerecently discovered putative par systems, which we delineate asthe type III and type IV systems, as this sets the stage for theensuing discussion on partition biology and Par protein structure–function relationships.

Type I and II par systems

The type I are the most numerous of the par systems and con-tain motor proteins with Walker-type or P-loop ATPase motifs[5]. These ATPase proteins are typically called ParA, and theircentromere-binding protein counterpart is called ParB. The type Ipar systems can be divided further into type Ia and type Ib onthe basis of the genetic organization of the par operon and thelimited sequence and size homologies of the Par proteins encodedby these systems (Figure 1B). In both type Ia and Ib systems,the gene for the Walker-type ATPase is located upstream of thatfor the centromere-binding protein. However, the location of thecentromere site differs: for the type Ia systems, the centromere-like site is located downstream of the par operon, whereas, in thetype Ib systems, the centromere is found upstream (Figure 1B).


Structure and function of plasmid partition proteins 3

The type Ia Par proteins are characteristically larger than type Ibproteins. Specifically, the type Ia ParA and ParB proteins typicallyconsist of 251–420 residues and 182–336 residues respectively.In contrast, type Ib ParA and ParB proteins generally contain208–227 residues and 46–113 residues respectively. Notably, thetype Ia ParA proteins have a dual role in partition; they function inboth partition and transcription autoregulation of their respectivepar operons [3–11,26–29]. Consistent with this additional role,type Ia ParA proteins contain an N-terminal region consistingof 108–130 residues in addition to their C-terminal Walker boxdomain. This N-terminal region contains a putative HTH (helix–turn–helix) DNA-binding motif, which is not found in the smallertype Ib Walker-box ATPase proteins. Thus, in the type Ib system,the motor proteins do not function as transcriptional regulators.However, maintenance of the correct cellular concentrationsof Par proteins is essential for proper partitioning by all parsystems. In the type Ib and type II par systems, the centromere-binding proteins carry out this important autoregulatory function(Figure 1B). There is no significant sequence identity or similaritybetween the type Ia and Ib centromere-binding proteins. Inaddition, type I centromere-binding proteins are sequentiallydiverse compared with the type II centromere-binding proteins.The most conserved region among the type Ia ParB proteinsis a predicted HTH motif. The type Ib centromere-bindingproteins display even more distinct sequences than the type IaParB proteins and contain no identifiable DNA-binding motif(s).Indeed, it was not until recent structures were solved that theDNA-binding motifs of these proteins were revealed.

The type Ia par loci from the E. coli P1 and F plasmids areamong the best characterized par systems [10,11,24,25]. TheP1 par system contains the prototypical ParA ATPase andthe centromere-binding protein ParB, whereas the F par systemencodes an ATPase called SopA and a centromere-binding proteinnamed SopB. Studies on these systems revealed that the ParB andSopB proteins bind to specific centromere sites and form higher-order complexes called partition complexes, which recruit theParA/SopA proteins for completion of the partition reaction [30–32]. More recently, Hayes and co-workers have carried out anextensive analysis of the type Ib par system from the SalmonellaNewport TP228 plasmid [33–35]. These studies revealed that theTP228 par system contains functional analogues of ParA andParB, called ParF and ParG respectively [33–35].

The second type of par system, termed type II, containsATPase proteins called ParM (for motor) that belong to theactin/hsp70 (heat-shock protein 70) superfamily. The centromere-binding proteins in type II systems are called ParR. The geneticorganization of type II systems is very similar to those of thetype Ib systems, wherein the parM gene is located upstreamof the parR gene and the centromere site is generally locatedupstream of the parMR genes (Figure 1B). The type II ParMproteins contain 236–336 residues, whereas the ParR proteins,similarly to the type Ib ParB proteins, are small, generally 46–120 residues in length [7,36–44]. Because the type II centromere-binding proteins also function in transcription autoregulation, theywere named ParR, for Par repressor. In contrast, the ParM proteinsshow no DNA-binding activity. Similarly to the type I centromere-binding proteins, the ParR proteins show little to no sequencesimilarity (or identity). Again, until structures were solved, theDNA-binding motif and manner of DNA recognition employedby these proteins was unknown.

The best investigated type II par system is that harboured on theE. coli R1 plasmid [36–44]. Like all type II systems, R1 containsa ParM ATPase and a ParR centromere-binding protein. The R1centromere, parC, is somewhat unusual in that it is discontinuous;it consists of two clusters of five 11 bp repeats, which are located

Figure 2 Representative par centromere sites

Well-characterized centromere-like sites are shown for two of each class of par systems (type Ia,type Ib and type II). The arrows represent repeat elements within each centromere. The arrowsare schematic; the lengths of the arrows do not represent nucleotide length. The number ofnucleotides in each repeat are indicated. For example, each sopC repeat is 43 bp, the P1 parSB-box elements are 6 bp (orange), and the A-box elements are 7 bp (blue). The central IHF siteis 29 bp (grey). The TP228 parH repeats contain 19 bp repeats, whereas the pTAR parSsite contains consensus repeats that are 7 bp. The type II pSK41 parC repeats are 20 bp,whereas the R1 repeats within parC are 11 bp in length.

on either side of the −10 and −35 DNA promoter motifs [13,40](Figure 2). The R1 par system has been thoroughly dissectedfrom a cellular, biological and biochemical standpoint. On thebasis of these detailed analyses, the insertional polymerizationmodel was formulated [45]. The foundation of this model isbased on the important discovery that the R1 ParM proteinforms filaments in an ATP-dependent manner [45]. Accordingto the model, ParM filaments are first captured between thepaired partition complexes, one end of each filament attachingto each partition complex. Continued filament growth betweenthe plasmids then forces the plasmids to the cell poles, henceleading to DNA segregation. Recent studies have demonstratedthat this process can be accomplished in vitro using only the threecomponents ParM, ParR and parC [46]. Thus this latter studyfirmly established that cellular factors are not essential for thegeneral mechanism of type II plasmid separation in vitro, althougha role for such factors in vivo cannot yet be excluded [46].

Double par loci on a single plasmid

The E. coli pB171 and Salmonella enterica R27 plasmids areunique in the plasmid par systems studied thus far in that bothcontain a type I and a type II par system [47–53] (Figure 1B). Todate, however, plasmids carrying two par loci of the same type(type I or II) have not been identified. Koonin [54] and Mushegianand Koonin [55] have speculated that all biological functionshave evolved independently at least twice and that analogousgenes can often replace each other in a process called non-ortho-logous gene displacement. Given the fact that type I systems aremore abundant and also more efficient than type II par systems inplasmid retention, Gerdes and co-workers have proposed that thetype I par systems, may in fact be replacing the type II systems[51]. The increased abundance of type I loci compared withtype II systems appears to support this idea. They cite the pB171plasmid as a possible evolutionary snapshot in non-orthologousgene displacement whereby the type I par locus is in the processof replacing the less efficient type II locus [51].

Type III and IV par systems

The majority of par systems can be categorized as either type I orII. However, recent data indicate that there are other types of parsystems. A possible addition to the family of plasmid par systems,which we shall term type III, was identified on the Bacillusthuringiensis virulence plasmid pBtoxis [56–58]. This putativenew par system encodes an upstream gene called tubR and adownstream gene, tubZ. Thus this genetic organization, which


4 M. A. Schumacher

places the gene encoding the centromere-binding protein beforethe motor protein, is distinct from the type I and II par systems[56]. A possible centromere site for the B. thuringiensis israelensissubspecies has been identified and consists of four 12 bp directrepeats [57]. The TubR protein, which contains a putative HTHdomain, was shown to bind specifically to these repeats. Perhapsthe most interesting feature of this system, however, was thefinding that the TubZ protein contains neither a deviant Walker boxmotif nor an actin-like fold and instead appears to be a member ofthe tubulin/FtsZ GTPase superfamily of motor proteins. In fact,it was clearly demonstrated that TubZ assembles into dynamicpolymers that exhibit directional polymerization with plus andminus ends, indicating that plasmid partition of pBtoxis may bemediated by tubulin-like polymers [56]. How TubZ might attach toplasmids to drive segregation remains unclear. However, it appearsthat TubR can recruit TubZ polymers to the partition site, similarlyto how the centromere-binding proteins of the type I and II parsystems engage their requisite ATPase motor proteins. The recentfinding that the Bacillus anthracis pXO1 plasmid RepX protein(now called TubZ-Ba) forms two stranded filaments identical withthose formed by the B. thuringiensis TubZ protein (TubZ-Bt)supports the contention that there may be several type III parsystems [58–60]. Indeed, at least four TubZ-like sequences havebeen found on different Bacillus plasmids [56].

Finally, a potential type IV partition system that utilizes onlyone protein has been identified on the Staphylococcus aureusplasmid pSK1 [61]. This protein, called Par, contains 245 residuesand was shown to be critical for extending the segregationalstability of the pSK1 plasmid, supporting the idea that it mayfunction in partition. It seems unlikely that a second protein ispresent in the pSK1 par operon as only one significant openreading frame, belonging to par, has been identified in the codingregion. How this protein may play both centromere-bindingand motor functions is unknown. However, structural predictionstudies suggest that Par contains an N-terminal HTH domain,which could function in centromere-binding, and a central coiled-coil domain, which might form polymeric structures similar tothose formed by coiled-coil-containing proteins in the cytoskele-ton of eukaryotic cells [62–64]. Therefore the pSK1 Par proteincontains domains that could conceivably perform both roles in par-tition. Clearly, more studies are needed to elucidate the mechan-ism of pSK1 segregation and the involvement of the pSK1 Parprotein in this process. Interestingly, Par homologues have beenfound on plasmids in a range of Gram-positive bacteria, includ-ing Staphylococcus, Streptococcus, Lactococcus, Lactobacillus,Clostridium and Tetragenococcus, suggesting that such systemsare not uncommon [65].

Bacterial chromosomal par systems

It is interesting that, although the mechanism(s) of DNA segreg-ation utilized by eukaryotes and plasmids have been delineatedin general terms, the means by which most bacteria partitionchromosomes is still largely a mystery. This is notably andsurprisingly true for the model Gram-negative organism, E. coli.Indeed, no partition proteins with homology with plasmid Parproteins have been identified in E. coli genome searches, nor haveany Par-like proteins been isolated from genetic screens [66].However, DNA segregation of some bacterial chromosomes doinvolve Par protein homologues and these proteins are surprisinglysimilar to their plasmid counterparts. The best-studied examplesof chromosomal par loci are from Bacillus subtilis, Caulobactercrescentus, Pseudomonas putida and Pseudomonas aeruginosa[67–70]. Although the present review focuses on studies onplasmid partition machinery, we shall briefly mention some of

these examples of bacterial chromosomal DNA segregation byplasmid-like Par proteins.

Bacterial chromosomal par systems appear to be hybrids ofplasmid Type Ia and Ib systems in that the chromosomal ParAproteins contain deviant Walker box motifs of the small Type Ibcategory, whereas the corresponding chromosomal centromere-binding proteins contain HTH motifs and are very similar toplasmid type Ia centromere-binding proteins [7]. Initial indi-cations that Par-like proteins might be involved in chromosomesegregation in bacteria came from genetic studies showing thatmutation of the chromosomally encoded B. subtilis ParB homo-logue, spo0J, resulted in a 100-fold increase in the production ofanucleate cells [71]. Found on the same operon as spo0J is agene, soj, which encodes a deviant Walker box-containing protein.Initial studies on Soj mutants suggested that it is not involvedin DNA segregation [71,72]. However, more recent data showthat Spo0J and Soj together play an important role in bringingtogether centromere sites that are far apart on the B. subtilischromosome and organizing the origin region into a compactstructure that facilitates separation of replicated origins [73]. Todo this, Spo0J, like its plasmid counterparts, binds a conservedDNA site, numerous copies of which are clustered about the soj–spo0J operon and the replicated chromosomal origins.

There are only a few cases in which chromosomal Par proteinshave been shown to be essential for bacterial DNA partition undernormal cellular conditions [74–76]. In C. crescentus, the ParB andParA homologues are required for chromosome segregation,and inactivation of either one is lethal. Another exampleis chromosome II of Vibrio cholerae. V. cholerae contains twochromosomes and each has a parAB system. Recent studies havefound that segregation of chromosome II is absolutely dependentupon its parAB system [77,78]. It is interesting to speculate thatthis chromosome may actually represent a co-opted plasmidthat has taken on a role as a host chromosome. In contrast withthese examples in which par systems are essential for DNAsegregation, some bacterial par systems are important only underspecialized conditions. For example, deletion of the Streptomycescoelicolor par system causes serious chromosome defects onlyduring the formation of chain spores [79]. Consistent with this,parAB transcription is greatly stimulated during S. coelicolorsporulation [79]. In P. putida, the parAB locus plays a vital rolein DNA segregation during the transition from exponential tostationary phase in cells grown in minimal medium [80,81]. Thusthese combined data suggest that plasmid Par homologues playimportant roles in DNA segregation in some bacteria, whereasthey may be important only under certain conditions in others.Consistent with this supposition, more than 50 homologues ofParA and ParB have been identified in bacteria thus far [82].

CELLULAR AND BIOCHEMICAL STUDIES REVEAL PLASMIDSEGREGATION STEPS

Cellular and biochemical studies have provided a general outlineof the steps involved in the plasmid partition process. Theimportant findings from these studies will be summarized nextto set the stage for the discussion of structural studies, which, inturn, have begun to elucidate the detailed molecular mechanismsinvolved in DNA segregation.

First step: formation of the partition complex

The first step of the partition reaction involves the co-operativebinding of the centromere-binding proteins to their cognate cen-tromere site. Just as eukaryotic centromeres serve as attachment



sites for spindle microtubules, plasmid segregation depends onthe creation of a defined nucleoprotein complex, called thepartition complex, which is formed by the accurate assembly ofcentromere-binding proteins on to the centromere site. As noted,little homology exists between centromere-binding proteins,and the centromere-like sites that they bind are also diverse.However, most plasmid centromere-like sites share an importantcharacteristic, which is that they consist of multiple DNA repeatelements (Figure 2). The repeats can be extensive, as exemplifiedby the F plasmid centromere, sopC, which contains 12 tandemrepeats of a 43 bp element [30,83]. Also, the parS centromere ofthe Agrobacterium tumefaciens pTAR plasmid contains 13 repeatsthat are separated by an integral number of turns of the DNAdouble helix [84]. The type II R1 parC centromere has a complexarrangement in that it consists of two sets of 11 bp repeats thatare separated by a 39 bp region that includes the parMR promoter[13]. But the most complex centromere is the parS centromereof the P1 plasmid. Similar, albeit less well studied, centromeresare found on related plasmids such as the E. coli P7 plasmid[85–88]. These centromeres contain a central binding site for theE. coli protein IHF (integration host factor) (Figure 2). IHF is anarchitectural protein that bends DNA by ∼180◦ [89]. On eitherside of the IHF site in the P1 centromere are asymmetricallyarranged DNA-binding motifs called A-boxes and B-boxes thatare recognized by the P1 ParB protein. Not only are the boxesasymmetrically arranged, they also differ in number on each armwhereby the ‘left’ arm (left of the IHF-binding site) containsone B-box followed by a single A-box, whereas the ‘right’ armcontains one B-box sandwiched between two A-boxes on theleft and one A-box on the right (Figure 2). Less complicatedcentromeres include the plasmid TP228 parH centromere, whichconsists of four direct repeats and the type II pSK41 centromere,parC, which also contains four tandem DNA repeats [35,90](Figure 2).

Despite the apparent diversity among centromeres andcentromere-binding proteins, in all cases it appears that theprotein–centromere interaction leads to the formation of higher-order partition complexes. The higher-order structure of thepartition complex is postulated to be important in the captureand activation of the motor protein for DNA segregation [90].The strongest evidence for higher-order protein–DNA structure,including DNA wrapping, in formation of the partition complexescomes from studies on the interactions between the centromere-binding proteins SopB and R1 ParR with their centromere sites.The dramatic effect on DNA topology caused by formationof the SopB–sopC partition complex is consistent with a structurein which the centromere DNA is wrapped in a right-handedmanner around a multimeric SopB protein core [91]. The R1parC centromere itself appears to be intrinsically bent, and ParRbinding leads to further distortion of the site [92]. Similarly, thecentromere of the Enterococcus faecium gentamicin-resistanceplasmid pGENT is also intrinsically curved [93]. In this regard,it is notable that analogous structural features are associated witheukaryotic centromeres. Specifically, studies indicate that thereis inherent curvature in Saccharomyces cerevisiae centromereDNA sites and binding of yeast centromere-binding proteins withthese DNA elements promotes DNA wrapping into higher-ordercomplexes [92–94]. These combined findings indicate that thespecific architecture of the higher-order partition complex playsa critical role in its function. In the case of partition systems, thefunction would probably be assembly of the motor protein to forma fully active segrosome. Indeed, as will be described, the recentstructure of the pSK41 ParR–centromere complex shows a highlywrapped protein–DNA superstructure, with overall dimensionsideal for ParM filament binding [90].

Recruitment of the motor protein is thought to take place afterthe two plasmids have paired. The pairing event is apparentlymediated by interactions between centromere-binding proteinsand/or centromere sites located on the partition complexes of thereplicated plasmids. Pairing was initially suggested by the findingthat plasmid centromeres act as incompatibility determinants.This means that plasmids that replicate or segregate using similarmechanisms cannot coexist in the same cell [95]. In contrast,plasmids with distinct partition systems are compatible, cancoexist and localize to alternative subcellular positions. Althoughpairing has been somewhat controversial, it has been demonstratedin vitro for the E. coli plasmids R1 and pB171 and in vivo for theP1 plasmid [8,96–98].

Second step: DNA separation as mediated by motor proteins

After plasmid pairing, the motor protein is recruited to create theactive segrosome and mediates plasmid separation. The mechan-ism(s) by which motor proteins carry out this feat was initiallyunclear, especially for the Walker-type ParA ATPase motor pro-teins. To be activated for partition, both type II ParM proteinsand type I Walker-type proteins must bind ATP. As noted, for thetype II ParM proteins, ATP binding was found to stimulate fila-mentation, thus providing an explanation for the ATP requirementand also providing a model for plasmid separation. Accordingto this model, growth of ParM filaments anchored between twoParR/parC-containing plasmids pushes or pulls the plasmidsapart. Interestingly, a recent study reported that GTP binding toParM stimulates the formation of filamentous structures identicalwith those formed in the presence of ATP [99]. Whether ATPor GTP, or a mixture of these nucleotides, functions in type IIpartition in vivo remains to be resolved.

The Walker-type ATPase proteins also require ATP bindingfor partition [26–28]. How ATP binding stimulates segregationby the type I motor proteins, however, has been less clear thanfor the type II motor proteins. Initial models, based primarilyon knowledge regarding other Walker-type proteins, suggestedthat type I motor proteins are monomers in the absence of ATPand form nucleotide sandwich dimers upon ATP binding [26].Dimerization is mediated via cross-contacts from one subunit tothe ATP molecule bound in the second subunit. However, it isdifficult to speculate how such a simple monomer to dimer switchcould cause the large-scale propulsion of paired plasmid DNAfrom the cell centre to opposite cell poles. More recent structuraland biochemical data, which have revealed that these Walker-type proteins can form polymers upon ATP binding, suggestthat they actually function in a manner similar to the ParMproteins in the type II par systems [3,6]. The ability of Walker-type proteins to form polymers is not unprecedented. Indeed, theWalker-type protein MinD forms polymers upon binding ATP.The MinD protein is part of the MinCDE system that is involvedin cell division control, and ATP binding to MinD causes therelease of its C-terminal region, which forms an amphipathichelix when localized to the cell membrane. Once attached tothe membrane, multiple MinD proteins coalesce and form longfilaments [100,101]. Along with MinC, the role of MinD is toprevent formation of the cell division septum at random locationsin E. coli, particularly near the cell poles. The inhibition of sep-tum formation by MinCD is caused by a rapid oscillation ofthis complex between the cell poles. This remarkable oscillationcorrelates with the polymerization of the MinD protein [101].

The theory that Par Walker-type proteins utilize filamentsformation for segregation has been somewhat controversial.However, data showing that several type Ia and Ib Walker-typeproteins can form polymers provide strong support for the idea that


6 M. A. Schumacher

this capability is important functionally. The pB171 ParA, whichis a member of the type Ib ParA family, was shown to both oscillateover the nucleoid and form polymers [52,102]. This led to a modelfor pB171 partition in which polymerization by pB171 ParApushes the plasmids apart by the insertion of ParA–ATP moleculesbetween the ParA–ParB/parC interface, similar to the insertionalpolymerization model proposed for type II segregation of theR1 plasmid [102]. However, an important feature of this model,called the ‘sweeping par model’ is that the force exerted by pB171ParA is assumed to be proportional to the number of filamentsinvolved in forming the polymer at that point. This model differsfrom the type II insertional polymerization model in some ways.Perhaps the major difference is that, unlike ParM, which formsnon-oscillating filaments that separate plasmids paired by ParRand pushes them to cell poles, oscillating pB171 ParA positionsplasmids near quarter cell locations. As a result, whereas thetype II R1 reaction is binary because the mitotic machinery canonly accommodate one pair of plasmids at a time, each attachedat the ends of the growing ParM filament, the pB171 ParA canposition many plasmids at a time because of the oscillating natureof its polymer, and so all plasmids present at a given momentcan participate in the segregation process. Notably, this modelis consistent with the observed higher efficiency of type I locicompared with type II. Another type Ib motor protein that has beenshown to form polymers is the TP228 ParF protein [34]. Similarlyto pB171 ParA, the polymerization of ParF is dependent on ATPand is stimulated by the presence of its centromere-binding protein[34].

The E. coli type Ia ParA protein SopA has also been shown toform filament-like structures [18,103,104]. Several detailed mech-anisms have been posited for SopA-mediated segregation basedon its ability to form polymers [103,104]. One model proposesthat SopA forms asters that project from the partition complex[103]. It is hypothesized that the asters not only function to pushthe plasmids apart, but also to position the plasmids at midcellbefore replication and maintain the position of daughter plasmidsafter separation. Thus, according to this model, the pushingand/or pulling forces that are imparted by the projecting asterswithin the cell would allow for the correct positioning of theplasmids in the cell. A second model, based on studies in whichthere was no evidence for aster formation, proposes that nucleoidDNA inhibits SopA polymerization. In this model, increased con-centrations of SopB bound to sopC alleviate the DNA inhibitionof SopA polymerization, allowing polymers to attach to SopB–sopC partition complexes [104]. Interestingly, in the studies usedto formulate the aster hypothesis, there was no evidence for DNAinhibition of SopA polymerization. A third study, which showsevidence for SopA oscillation between cell poles, suggests thatSopA filaments function as a railway track for the movement ofthe plasmids [18]. Despite the differences in detail, all of thesemodels propose that F plasmid segregation is powered by SopApolymers, which are formed in a manner stimulated by ATP andSopB/sopC.

STRUCTURAL STUDIES ON PARTITION PROTEINS

Cellular and biochemical studies have provided a general outlineof the plasmid partition process. However, multiple questionsregarding the structure–function relationship of Par moleculesremain and must be addressed in order to provide a complete anddetailed understanding of the mechanisms employed by the Parproteins to segregate DNA. Outstanding questions regarding thefirst step include: what types of DNA-binding motifs do centro-mere-binding proteins utilize; how do these motifs recognize

centromere repeats; how do multiple centromere-binding proteinsbind co-operatively to multiple repeats to form a higher-orderpartition complexes; and what kind of structures are adopted bythese higher-order complexes? In terms of the second step ofplasmid partition, important questions include: what structuresare adopted by the putative Walker-type proteins (do they reallycontain canonical Walker-type folds?); how does ATP bindinglead to a ‘switch’ in conformational states that ultimately resultsin plasmid separation; how does interaction with the partitioncomplex stabilize and possibly stimulate this process; and whatrole does ATP hydrolysis play in plasmid separation? As discussedbelow, recent structural information has started to shed light onsome of these questions.

Centromere-binding proteins: sequence diversity beliesstructural homology

Within the last year, new structures have been obtained for severalcentromere-binding proteins. As a result, structures are nowavailable for each type of centromere-binding protein: type Ia,type Ib and type II. Remarkably, these combined structures indi-cate that, despite the significant lack of sequence homologyamong these proteins, they share common DNA-binding folds.In fact, it appears that there are two primary structural classes ofcentromere-binding proteins: those that contain HTH folds, whichare of the type Ia family, and those that contain RHH (ribbon–helix–helix) DNA-binding motifs, which unexpectedly includeboth the type Ib and type II centromere-binding proteins.

Structures of type Ib centromere-binding proteins

The type Ib centromere-binding proteins are typically small,consisting of 46–113 residues. The sequences of these proteinsprovided no hint as to how centromere binding is mediated.However, recent structures of these proteins and their complexeswith DNA have revealed the DNA-binding motif they utilize andhow they bind DNA (Figure 3).

Structure of TP228 ParG protein

One of the best-characterized type Ib par systems is that ofthe E. coli multidrug-resistance plasmid, TP228. The TP228centromere-binding protein, called ParG, is a 76 residue, 8.6 kDadimer that is essential for TP228 plasmid retention. The structureof apo ParG was solved by NMR and revealed that it contains abipartite structure with an N-terminal disordered region (residues1–32) and a C-terminal region (residues 33–76) that has a RHHDNA-binding motif [105] (Figure 3B). The RHH topology isβ1, residues 34–41; α1, residues 43–55; α2, residues 60–75.The RHH is a very common DNA-binding motif present in theArc/MetJ superfamily of DNA-binding proteins [106]. In fact, itis predicted that there are over 2000 RHH-containing proteinsin various bacterial sources, 55 in bacteriophages and 300 suchproteins in archaea [106]. Thus far, the RHH fold appears tobe absent from eukaryotes. However, RHH folds are difficultto predict without experimental data as sequence conservationis strikingly low among these proteins and there is a generallack of strictly conserved residues within the fold. As was noted,this was the case for the centromere-binding proteins. Furtherimpeding the identification of RHH proteins is the fact theyregulate multiple diverse cell processes such as transcription, thelytic cycle of bacteriophages and cell division [106]. However, allRHH proteins whose structures have been solved to date formdimers-of-dimers upon DNA binding, whereby each β-strandinserts into consecutive major grooves. The defining characteristicof the RHH motif is the utilization of β-strand residues for making



Figure 3 Structures of type Ib centromere-binding proteins

(A) Schematic diagram showing the domain structure of the type Ib centromere-binding proteins. The N-terminal domain (grey) is a flexible region that functions in ATPase stimulation, ATPaserecruitment and pairing. In addition, a role in DNA binding has been suggested. The C-terminal domain (magenta) contains the DNA-binding RHH motif. In some cases, there is a short regionfollowing the RHH (shown in grey). The RHH motifs of the structures in (B) and (C) are shown in the same orientations for comparison purposes. (B) Structure of the ParG protein from the E. coliTP228 plasmid shown colour-coded as in (A) [105]. The protein is a dimer. The N-terminal flexible region is grey, and the RHH, which mediates all the dimer contacts, is magenta. (C) Structure ofStreptococcus pyogenes plasmid pSM19035 ω protein colour-coded as in (A) [110,111]. On the left is the apo structure, and on the right is the ω–DNA structure. The DNA is shown as sticks andis coloured blue. N- and C-termini regions are labelled for one subunit in each apo structure. (B) and (C) were modelled using PyMOL (DeLano Scientific).

base-specific DNA contacts. Therefore it is likely that the RHHfold of ParG binds its DNA centromere-like site by forming adimer-of-dimers and inserting its double-stranded β-sheet intothe DNA major groove.

In addition to binding their centromere sites to form higher-order partition complexes that recruit and stabilize motor proteins,centromere-binding proteins also stimulate the ATPase activity ofthe motor protein. This presents a conundrum as ATP hydrolysisleads to depolymerization of ATPase motor proteins. To date, itis not understood at a molecular level how these two opposingfunctions are mediated and co-ordinated to achieve accurateplasmid separation and this remains one of the important questionsin partition biology. In studies aimed to address this issue, Hayesand co-workers have shown that there are two separate regionswithin the flexible N-terminal region of ParG that mediate theseactivities: one acts to promote polymerization of the Walker-box protein ParF and the other to enhance its ATPase activity30-fold [107]. Specifically, they identified residue Arg19 as partof a putative arginine finger motif within the N-terminal region ofParG that is responsible for stimulation of the ParF ATPaseactivity, but which is dispensable for the ParG-mediated affectson ParF polymerization. Arginine fingers are common motifsutilized by P-loop NTPases, either in cis, where it is from the sameprotein, or trans, where, similar to the case of ParG and ParF, it islocated on another protein [108]. These motifs, which are typicallyfound on extended loop regions on a protein, stimulate nucleotidehydrolysis by inserting into the active site and stabilizing thetransition state. Because ATPase-interacting regions located ontype Ia and Ib centromere-binding proteins are usually locatedin extended regions and contain arginine residues, an attractive

hypothesis, proposed by Hayes and co-workers, is that all theseproteins contain arginine finger motifs that are used in transto enhance the ATPase activity of their requisite ATPase motorproteins [107].

Remarkably, two additional functions have been posited forthe N-terminal domains of type Ib proteins: DNA binding andpairing. Specifically, studies on ParG indicate that residues inits N-terminal region can affect how it organizes the formationof higher-order ParG–DNA structures [109]. Moreover, the con-tinued deletion of this N-terminal tail greatly perturbs its DNA-binding kinetics [109]. Additionally, studies on the ω proteinfrom the Streptococcus pyogenes plasmid pSM1903, anothertype Ib centromere-binding protein with a RHH fold, revealedthat removal of its entire N-terminal tail causes a 2-fold reductionin its DNA-binding affinity [110]. Indications that the N-terminalregion of type Ib centromere-binding proteins impact pairingcome from studies on the pB171 ParB protein, which showed thatremoval of its N-terminal tail abrogated ParB–ParB interactionsbetween plasmids [53]. Combined, these data demonstrate that theflexible N-terminal regions of type Ib centromere-binding proteinsplay several pivotal roles in partition (Figure 3A). However, theprecise locations of these functional regions within the N-terminaldomains of these proteins have yet to be mapped, and their specificmolecular mechanisms have yet to be resolved.

Structure of inc18 ω protein in the presence and absence of DNA

In addition to ParG, the structure of the ω protein from theStreptococcus pyogenes plasmid pSM19035 found in the Gram-positive broad host range inc18 family of plasmids has also been


8 M. A. Schumacher

determined [110,111]. Like most type Ib centromere-bindingproteins, ω functions in transcriptional autoregulation. On theother hand, ω is distinct from other type Ib centromere-bindingproteins in that it also acts as a global regulator of transcription[111]. To carry out these functions, ω binds co-operatively to DNAsites containing seven to ten consecutive non-palindromicDNA heptad repeats (5′-A/TATCACA/T-3′). Interestingly, thesesites can be arranged as direct or inverted repeats. ω binds withlow affinity (Kd of >500 nM) to one heptad, but it binds stronglyto DNA sites containing two or more consecutive heptad repeats[110,111]. The heptad arrangement also affects the affinity of ωfor the DNA site, with direct or converging repeats providing thestrongest binding interaction.

Structural and biochemical studies on the Streptococcuspyogenes ω protein revealed that, similarly to ParG, it has abipartite structure with an N-terminal flexible region and a C-terminal domain containing a RHH motif (Figure 3C). Theflexibility of the N-terminal region is reflected in the structureof apo ω, whereby the N-terminal 20 amino acids were lost toproteolysis during crystallization [111]. The C-terminal RHH hasthe topology of β, residues 28–32; α1, residues 34–47; and α2,residues 51–66. To gain insight into the unusual DNA-bindingpreferences exhibited by ω, structures were solved in the presenceof direct and converging 17 bp DNA repeats using a deletionmutant of the ω protein, �19ω, in which the first 19 residues wereremoved. As noted, the N-terminal region has been implicated tobe important in DNA binding, and this deletion results in a 2-fold reduction in DNA binding compared with wild-type protein[110,111]. However, the role(s) played by the N-terminal tailsof type Ib centromere-binding proteins in DNA binding has yetto be determined as, so far, the �19ω–DNA complexes are theonly structures available for a type Ib centromere-binding proteinbound to cognate DNA.

The ω–DNA structures show that ω binds its DNA as adimer-of-dimers, wherein each dimer binds a consecutive DNAmajor groove. Unexpectedly, the crystallographic asymmetricunit contains a �19ω dimer-of-dimers bound to a 17 bp DNAsite, which in turn is arranged pseudocontinuously with a seconduncomplexed 17 bp DNA site. As a result, there is an unbound17 bp DNA site on either side of each ω–DNA complex in thecrystal lattice (Figure 3C). The DNA bound by ω is essentiallystraight and B-DNA in character. The presence of the uncom-plexed operator in the structure permitted the direct comparison ofprotein-bound and protein-free DNA conformations. This analysisshowed that the unbound DNA adopts the same conformationas that bound by �19ω. Therefore any deviations from idealB-DNA observed in the structure of the �19ω-bound DNAare the likely result of nucleotide sequence-induced alterations.Comparison of the structures of ω bound to direct and convergingrepeats showed that the protein can bind these different sitesbecause the contacts of ω to the two DNA sites are related by apseudo-2-fold rotation axis, and, as a result, the specific protein–DNA interactions are comparable, despite the heptad orientation[110]. Presumably, the formation of the higher-order ω–DNApartition complex involves the co-operative interaction of multipleω dimers-of-dimers binding to consecutive DNA heptad repeats.However, confirmation of this awaits structural studies on ω andother type Ib centomere-binding proteins bound to full-lengthcentromere sites.

Structures of type II ParR proteins

Structures for type II ParR centromere-binding proteins haveonly become available within the last year. Similarly to the

type Ib centromere-binding proteins, the amino acid sequencesof the type II centromere-binding proteins revealed no clues aboutthe structures these proteins adopt. However, these proteins aresimilar in size to the type Ib centromere-binding proteins. Thus itwas intriguing when the structures of the pB171 and the pSK41type II ParR proteins were solved and shown to belong to thesame family of DNA-binding proteins as the type Ib centromere-binding proteins, namely the RHH family (Figure 4).

Structure of pB171 ParR protein

The E. coli pB171 ParR protein functions as the centromere-binding protein for the type II locus on the pB171 plasmid, whichcontain both type I and II par systems [48–53]. The structureof the full-length pB171 ParR protein was recently solved inthe absence of DNA [112]. The C-terminal 35 residues were notresolved in this structure, neither were residues 1–5. The structure,consisting of residues 6–95, revealed a RHH motif and a C-terminal domain composed of a three helix ‘cap’ (Figure 4B).The topology is β1, residues 7–14; α1, residues 20–30; α2,residues 36–52; α3, residues 56–65; α4, residues 71–81; andα5, residues 88–94. The three-helix cap at the C-terminal regionnot only reinforces the tight dimerization of the RHH, but alsostabilizes interactions between dimers-of-dimers. Several RHHproteins contain domains outside the RHH motif that similarlyact to bolster the dimer and dimer-of-dimer; however, none ofthese structures is similar to the pB171 three-helix cap. A notablefinding in the crystal structure was the creation in the lattice of acontinuous helical structure created by the assembly of 12 ParRdimers per turn. In this arrangement, the N-terminal RHH motifsface one direction, whereas the C-termini point in the oppositedirection. This was interesting because EM (electron microscopy)images of the R1 ParR protein bound to its centromere site parC,revealed that the ParR–parC partition complex forms ring-likestructures on pre-linearized DNA that are between 15 and 20 nmin diameter [112]. Because R1 and pB171 ParR proteins probablyform similar higher-order partition complexes, it was postulatedthat the EM projections of R1 ParR–parC were possibly hidingthe helical nature of the arcs observed in the pB171 ParR apostructure. However, it was not possible to distinguish the exactnature of the superstructure formed by the R1 or pB171 ParR–DNA partition complexes by these data. The resolution to theissue was obtained by the crystal structure of the type II pSK41ParR–centromere complex, which revealed the first structure of ahigher-order partition complex [90].

pSK41–ParR–centromere complex reveals partition complex structure

pSK41 is a prototypical multiresistance plasmid from Staphyloc-occus aureus and is stably maintained by the presence of a type IIpartition system encoding ParR and ParM proteins [113]. ThepSK41 Par proteins show little sequence homology with R1ParR and ParM. Nonetheless, the pSK41 ParM protein is readilyidentifiable as an actin-like protein, thus establishing the systemas a type II par system. The pSK41 centromere site was identi-fied by a combination of footprinting and FP (fluorescencepolarization)-based DNA-binding studies [90]. These analysesrevealed that the centromere consists of four 20 bp elements,in which the 20 bp repeat constitutes the minimal element forbinding of a ParR molecule (Figure 2). The simplicity of thiscentromere site made it an ideal candidate for attempts to obtainthe structure of a higher-order partition complex. As data-qualitycrystals of the pSK41-full-length ParR–centromere complexcould not be obtained, the protein was subjected to limited



Figure 4 Structures of type II centromere-binding proteins

(A) Schematic diagram showing the domain structure of the type II centromere-binding proteins. The N-terminal domain (magenta) forms a RHH domain responsible for centromere binding. TheC-terminal region is a partially flexible domain that functions in ParM binding and also contributes to the stabilization of the RHH dimer and dimer-of-dimers. The RHH motifs of the structures in(B) and (C) are shown in the same orientations for comparison purposes. (B) Structure of the E. coli pB171 ParR colour-coded as in (A) [112]. The N-terminal RHH domain is coloured magenta,and the C-terminal region, which consists of a three-helix cap and additional disordered residues, is grey. N- and C-terminal regions are labelled. (C) Structure of the Staphylococcus aureus pSK41ParR protein coloured as in (A) [90]. On the left is the protein in the absence of DNA, and on the right is the ParR–centromere complex. N- and C-termini regions of one subunit are labelled in theleft-hand panel. The DNA is shown as sticks and coloured blue. (B) and (C) were modelled using PyMOL (DeLano Scientific). An interactive three-dimensional version of the structure shown in(C) can be seen at http://www.BiochemJ.org/412/0001/bj4120001add.htm.

proteolysis, and the minimal DNA-binding domain was foundto reside in the first 53 residues of the 109 residue protein. Thisdomain, termed ParRN, contains all of the determinants requiredfor centromere binding as ascertained by FP-binding studies[90]. The structure of the ParRN-centromere was determined byutilizing a 20 bp repeat, which stacked pseudocontinuously in thecrystal to form the full-length centromere (Figure 4C).

The structure revealed that, like pB171 ParR, pSK41 ParRcontains a RHH motif in its N-terminal DNA-binding domainwith topology of β1, residues 5–12; α1, residues 16–25; and α2,residues 33–47. Like all RHH proteins, the β-strands combinein an antiparallel fashion and helices interdigitate to form theextensive RHH dimer. The DNA elements bound by each ParRdimer-of-dimer stack pseudocontinuously to generate the full-length centromere. Thus the most notable feature of the structureis the creation of the higher-order partition complex that mediatesfull segrosome assembly (Figure 4C). Consistent with the co-operative nature of ParR binding, the protein dimer-of-dimers thatbind the DNA form intimate protein interactions that lead to thegeneration of a continuous protein superstructure that has distinctpositive and negative electrostatic surfaces. As would be expected,the positive face wraps the centrosomal DNA about itself tocreate the unique superstructure that is best described as a largesuperhelix. Characteristic of the superstructure is its superhelicalparameters: it has a pitch of ∼24 nm with six ParR dimers-of-dimers in one turn of the superhelix and a measured diameter of∼18 nm. Within this structure, the C-terminal regions of the ParRprotein would face inwards, towards the pore, which suggests thatit plays a role in ParM recruitment. This was confirmed by bindingexperiments, which showed that ParM bound only to complexescontaining full-length ParR and not ParRN [90].

DNase I-protection studies carried out on the pSK41 ParRbinding to the centromere revealed striking nucleosome-likeperiodicity in the protection pattern consistent with the crystalstructure. As would be expected from binding a continuous proteinsuperstructure, the footprint showed almost complete protection.Indeed, only eight symmetrically disposed adenine nucleotideson each strand are susceptible to DNase I attack. The basis forthe susceptibility of these nucleotides is revealed by the crystalstructure, which shows that these adenines reside in the onlyminor groove positions that are exposed outside the partitioncomplex superhelix and between ParR molecules that bind eachrepeat (Figure 4C). Thus the multiple features of the DNase Ifootprint are readily explained by the crystal structure. In thissame study, cryo-EM was also carried out whereby the partitioncomplex formed by binding of ParR to the centromere site withno surrounding DNA regions was examined. The image revealeda circular superhelical structure with dimensions essentiallyidentical with those observed in the crystal structure.

The structural studies on the pSK41 ParR–centromere complexresolved the issue of whether R1 and pB171 ParR–centomerecomplexes formed rings or helices as, clearly, the structure is asuperhelix [90,112]. Indeed, the measured diameter of the pSK41superhelical partition complex crystal structure was ∼18 nm,which closely matches the 15 and 20 nm diameter obtained forthe superhelical rings of the R1 ParR–centromere complex byEM [112]. These data, which indicate a conserved structureof the type II segrosome assembly, seem to support the notionthat the higher-order structure of the partition complex is likelyto be crucial to its function of recruitment of ParM filaments andtheir stabilization. Indeed, as discussed below, recent EM data onParM filaments combined with the structure of the pSK41 partition


10 M. A. Schumacher

Figure 5 Structures of type Ia centromere-binding proteins

(A) Schematic diagram showing the domain structure of the type Ia centromere-binding proteins. The N-terminal domain (grey) is a flexible region involved in ATPase activation and recruitment.Motif B (cyan) functions as a higher-order oligomerization domain. The HTH unit (magenta) is a three-helix unit containing an HTH motif. This unit is highly conserved in the type Ia structuressolved to date. The linker domain (blue) is a helical domain that links the HTH unit to the dimer domain. The dimer domain (light grey) mediates dimerization and is not conserved among the type Iacentromere-binding proteins. The HTH units of each structure are shown in the same orientation with its three helices labelled H1–H3 (magenta). (B) Structure of the T. thermophilus Spo0J proteincolour-coded as in (A) [114]. Although included in crystallization, the N-terminal domain was not visible. Motif B (cyan) mediates dimerization between Spo0J subunits at high concentrations. TheHTH unit is magenta and the linker domain is blue. N- and C-termini regions are labelled. (C) Structure of the P1 ParB-(142–333)–parS small complex [123]. The HTH unit is shown as the sameorientation as that of Spo0J and is coloured magenta. The linker domain is blue, the dimer domain is grey, and the N- and C-termini regions of one subunit are labelled. The DNA is shown as asurface and is coloured yellow. (D) Structure of the RP4 ParB homologue, KorB. The C-terminal dimer-domain (residues 297–358) and DNA-binding domains (residues 101–294) were solved asseparate domains (not all residues were visible) [118,119]. The KorB dimer was modelled by linking the two domains by a flexible region (indicated by broken lines). Part of motif B present in thestructure is coloured cyan. The HTH unit is magenta and the linker domain is blue. The dimer domain, solved separately, is grey and is shown as linked by flexible residues of the linker domain.The DNA is shown as a yellow surface. (B)–(D) were modelled using PyMOL (DeLano Scientific).

complex provide insight into the role played by the partition com-plex in ParM filament recruitment and final segrosome assembly.

Structure of type Ia ParB centromere-binding proteins

It had been predicted that the type Ia ParB proteins containHTH motifs. Studies on the type Ia centromere-binding protein,SopB, also clearly showed that these proteins form higher-ordersegrosomal structures. The DNA-binding properties of theseproteins that enable such higher-order structure formation arestarting to be resolved with recent structure determinations oftype Ia centromere-binding proteins.

All plasmid centromere-binding proteins share in commonthe fact that they are multidomain proteins that contain flexibleextended regions, usually at their N- and/or C-termini. Yet, thetype Ia centromere-binding proteins clearly differ from those inthe type Ib and II families. The type Ia proteins are larger than thetype Ib and II proteins and typically contain between 182 and336 residues. Sequence comparisons indicate that the type Iacentromere-binding proteins have two conserved regions, whichcorrespond to a domain called motif B and a HTH unit. Motif B isnear the N-terminal region and the HTH unit is centrally located(Figure 5A). The linker domains of type Ia proteins show limitedsequence and, as it turns out, structural homology (Figure 5A).Like the type Ib and II centromere-binding proteins, all the type IaParB proteins that have been characterized form dimers whereby

the dimer domain is located in the C-terminal ∼70 residues. TheC-terminal dimer domain is one of the least conserved domainsamong type Ia members. As will be described, this is reflected inthe finding that this domain can adopt very different structures.In fact, in one case, this domain functions in DNA binding as wellas dimerization.

Structure of Thermus thermophilus chromosomal protein Spo0J

The first described structure of a type Ia centromere-bindingprotein was that of the N-terminal domain of the T. thermophilusSpo0J, which is a chromosomally encoded type Ia protein [114].Attempts to crystallize the Spo0J protein from B. subtilis andeight other bacterial genomes all failed. Only a truncated formconsisting of residues 1–222 of the 269 residue T. thermophilusSpo0J was successfully crystallized. This fragment contains theregions of highest homology among ParB proteins, motif B andthe HTH unit. Spo0J homologues typically bind DNA sitescontaining multiple 16 bp repeats located in the origin proximalregion of the requisite bacterial chromosome [69,115]. Gel-retardation assays revealed that the Spo0J-(1–222) fragment isgreatly weakened for DNA binding, despite the fact that it containsthe HTH unit [114]. This is consistent with data on other type IaParB proteins, which indicate that the C-terminal dimer domainis essential for high-affinity DNA binding. In most cases, thisdomain is not directly involved in DNA binding by type Iacentromere-binding proteins and thus it appears that dimerization



is important in mediating the proper docking of the HTH units onto successive grooves of the DNA.

The structure of Spo0J-(1–222) is predominantly helical(Figure 5B). Residues 1–22 and 210–222 were not visible,consistent with data indicating that the N-terminal domains oftype Ia proteins are flexible. The structure can be broken into threemain regions based on homology with other type Ia proteins. Theregion containing residues 23–118 corresponds to the motif B re-gion and consists of four α-helices and a two-stranded β-sheet,with topology α1–α2–α3–β1–β2–α4 (Figure 5B). The HTH unitincludes residues 119–160. The linker domain, which in the full-length protein connects to the dimer domain, includes residues161–209. Although Spo0J-(1–222) does not contain the dimerdomain, a clear dimer is observed in the crystal structure, whichis formed by contacts primarily involving residues from motif B.There are two main interaction interfaces within the dimer: onebetween residues from α1 of one subunit and residues from α3,β1 and β2 of the other subunit and the second between α2 of onesubunit and helices 4, 5, 8 and 10 of the other subunit. Despitethe large surface area buried in this dimer interface, biochemicaldata show that Spo0J-(1–222) only dimerizes when the proteinis at high concentrations. This is consistent with studies on othertype Ia ParB proteins, such as P1 ParB, which suggest that motifB is involved in secondary oligomerization when bound to DNA[86].

Structures of RP4 KorB

The structures described above underscore a conserved feature ofcentromere-binding proteins, their flexible multidomain nature.Following this general theme, the type Ia RP4 KorB protein hadto be broken into domains in order for structures to be obtained.KorB is carried on the RP4 plasmid and is a member of theE. coli incompatibility group P (IncP-1α) [116,117]. This plasmiddisplays a broad host range and thus can be transferred to avariety of Gram-negative bacteria [117]. Structures have beensolved for the N-terminal HTH-containing domain of KorB aswell as the C-terminal dimer domain [118,119]. The KorB C-terminal dimer domain (residues 297–358) consists of a five-stranded antiparallel all-β structure with striking homology withthe SH3 (Src homology 3) domain found in eukaryotic signallingproteins (Figure 5D) [120,121]. However, unlike the SH3 domain,the KorB C-domain is missing a long loop between β1 and β2that is essential for the SH3 recognition of proline-rich motifs. Incontrast, the KorB C-domain contains a significantly elongatedβ5 compared with SH3 domain structures and is essential forKorB dimerization.

The KorB C-terminal domain is not directly involved incentromere binding. Instead, this function is fulfilled by theN-terminal region of KorB [119]. However, similarly to Spo0J,the C-terminal domain of KorB is critical for high-affinity DNAbinding by KorB again probably by correctly positioning andanchoring the DNA-binding domains on cognate DNA. Insightinto how KorB binds its individual centromere-like palindromicrepeats was provided by the structure determination of the KorBDNA-binding domain (residues 101–294) bound to one of its12 operator/centromere-like sites, Ob [120]. The structure of theKorB DNA-binding domain consists of eight helices, includ-ing the C-terminal portion of motif B (coloured cyan inFigure 5D), the DNA-binding HTH unit (magenta) and part ofthe linker region (blue) connecting the HTH unit to the dimerdomain (Figure 5D). Each half site of the palindromic operatorsite is bound by one KorB subunit in its major groove (Figure 5D).Although the KorB HTH has a canonical HTH structure, Thr211

and Arg240, which are outside the recognition helix and located

Figure 6 Structure of the P1 ParB-(142–333)–parS double B-boxinteraction: P1 ParB is a bridging factor

(A) Sequence of the P1 parS centromere site. The site is divided into right and left ParB-bindingsites, with an IHF site located in the centre. The A- and B-boxes are labelled. The leftside B1and A1 boxes are coloured blue and green, whereas the rightside A2, A3 and B2 boxes arecoloured yellow, green and blue respectively. Note that the third A-box, A4, which is located tothe left of B2, is not necessary for partition and is not shown. The rightside parS site, called parSsmall, encompasses A2–A3–B2 and is the minimal site required for partition. (B) Structure ofP1 ParB-(142–333) bound to an A3–B2-containing DNA site in which the protein dimer–DNAduplex stoichiometry was 1:2 [127]. One ParB-(142–333) subunit is coloured magenta and theother is yellow. The DNA is shown as surfaces with box elements coloured as in (A). Modelledusing PyMOL (DeLano Scientific). An interactive three-dimensional version of the structureshown in (B) can be seen at http://www.BiochemJ.org/412/0001/bj4120001add.htm.

on the second and fourth helices in the linker domain, are utilizedfor base-specific contacts.

Structures of P1 ParB–centromere complexes

As noted, the E. coli P1 and F par loci were the first partitionsystems to be discovered and have since served as paradigmsfor studies on plasmid partition [4,10,12,16–17,24–29,85–88].The P1 par system is of particular interest owing to its unusualcentromere-like site. Studies carried out largely in the Funnell andAustin laboratories have provided a detailed description of the P1centromere site and how it is recognized by the P1 ParB protein[24,25,85–88]. These studies showed that the P1 centromere site,parS, is an unusual ∼74 bp DNA centromere element that canbe divided into three main regions: a centralized ∼29 bp bindingsite for IHF, a so-called ‘rightside’ region with two heptamericA-boxes, A2 and A3 (the third A-box, A4, to the right of B2is not essential for partition) and one hexameric B-box, and a‘leftside’ element with one A-box and one B-box (Figures 2 and6A). Although not required for partition, the E. coli IHF αβheterodimer increases partition efficiency by bending the centralregion of parS to juxtapose the A-box- and B-box-containing


12 M. A. Schumacher

‘arms’, which appears to facilitate ParB binding across the bend(Figure 5C) [122].

Interestingly, data revealed that both the A-boxes and B-boxes are recognized and bound by P1 ParB [86]. This findingdistinguishes ParB from other centromere-binding proteins,which only bind one cognate DNA element. Although full-length parS provides maximal partition efficiency, the rightsideparS site composed of A2–A3–B2 is sufficient for partition inan IHF-independent manner. P1 ParB residues 142–333, whichcontains the HTH unit, linker domain and dimer domain, containall the determinants required for centromere binding. Similarlyto other type Ia centromere-binding proteins, motif B functionsas a secondary oligomerization domain in P1 ParB when theprotein is present at high local concentrations. The extreme N-terminal region of P1 ParB and nearly all other type Ia centomere-binding proteins are used to bind to their requisite motor proteins(Figure 5A). An exception is KorB, which binds its motor proteinusing residues within its centromere-binding domain [86,116].

Insight into how ParB can recognize two DNA elementsand bind the unusual parS centromere site were provided byrecent structures of ParB-(142–333)–rightside parS complexes(Figure 5C) [123]. These structures show that ParB-(142–333)comprises two separate domains. The first corresponds to residues147–270 and includes the HTH unit and linker region. Thisdomain consists of seven α-helices and is connected by a shortflexible linker (residues 271–274) to the C-terminal domain(residues 275–333). As in other type Ia centromere-bindingproteins, the C-terminal domain mediates dimerization. UnlikeKorB, which contains a C-terminal SH3-like domain, the C-terminal domain of P1 ParB contains a novel fold consisting ofthree antiparallel β strands and a C-terminal α helix [118,123].These elements lock together in the dimer to form a continuousantiparallel β-sheet, flanked by a coiled-coil (Figure 5C). Uniquestructural features evident in the ParB–DNA structures explainhow ParB can interact with multiple arrays of box elements onthe looped parS site [123]. Importantly, each box element isbound by a separate DNA-binding module: the A-boxes are boundby the HTH motif, whereas the B-boxes are recognized by thedimer domain. In addition, the structure shows that ParB acts as abridging factor between DNA boxes, explaining how it can bindsites on looped DNA or box elements on adjacent plasmids.

Importantly, the two DNA-binding modules of ParB do notinteract with each other and freely rotate, enabling them to contactmultiple arrangements of A- and B-boxes, as found in parS(Figure 5C). Residues from the recognition helix of the HTHcontact the major groove of the A-box elements. Interestingly,only one of these base contacts appears to be specific, suggestingthe possibility that the HTH domain may play a role in non-specific DNA spreading. Indeed, non-specific binding of P1ParB to DNA enables it to bind several kilobases upstream anddownstream of the parS site [124–126]. Studies on P1 ParB andSopB, which both show spreading, suggest that DNA spreadingmay be a shared feature of at least some of the centromere-binding proteins [104,115,124–126]. Initial data on spreading-defective ParB mutants led to the speculation that it was importantfor partition. However, more recent studies in which plasmidstability was determined after spreading was restricted by theintroduction of roadblocks on either side of parS revealed thatextensive spreading was, in fact, not required for plasmid partition[124]. One result of spreading is the silencing of genes severalor many kilobases away from the ParB–DNA interaction site[125]. The exact role(s) that spreading plays in DNA partitionand transcription regulation has yet to be clarified.

Unlike the HTH domain–A-box interaction, the contacts madeby the novel dimer domain to the B-box are highly sequence-

specific. Initial structures of ParB-(142–333)–parS rightsideshowed only one side of the dimer domain bound to a B-box.However, this was due to the stoichiometry of box elements toParB dimer used in the initial structural studies. Indeed, modellingsuggested that each side of the dimer domain should be capable ofB-box binding. The more recent structure of ParB-(142–333)bound to a 16 bp A-box/B-box-containing site confirmed this pre-diction and not only revealed a completely novel type of protein–DNA interaction, but also indicated that one P1 ParB is ableto interact simultaneously with four separate DNA duplexes orDNA sites on bent DNA or a mixture of these permutations[127] (Figure 6). This multi-bridging capability has importantimplications for the ability of P1 ParB to form wrapped nucleo-protein structures as well as mediate plasmid pairing. Indeed,the unique DNA-bridging function of ParB reveals how it canbind across the rightside and leftside arms of the bent parSsite. This unusual bridging capability would also permit P1 ParBto bind between the two arms of one looped parS site whilesimultaneously contacting a parS site on a second plasmid, thusproviding a very attractive model for plasmid pairing. Moreover,the multi-bridging capability, especially that provided by thedouble B-box interaction, which juxtaposes DNA close in space,suggests multiple strategies for forming higher-order partitioncomplex superstructures by P1 ParB.

Comparison of the Spo0J, KorB and ParB structures suggestthat there are larger differences among the type Ia proteins ascompared with the type Ib and II centromere-binding proteins.Although a mechanism for partition complex formation similarto that demonstrated by pSK41 ParR can be envisioned for allcentromere-binding RHH proteins, a clear structural model for thepartition complex by the type Ia proteins is not yet forthcoming.

Structures of motor proteins: mechanisms of plasmid separation

Once the higher-order partition complex is formed, the final stepin partition is formation of the final segrosome via recruitmentof the motor protein, which is typically either an actin-likeprotein (type II) or, more commonly, a Walker-type protein(type I). Filament growth then propels the plasmids to oppositecell poles. Currently, structural information is available for onetype II plasmid motor protein, ParM from the E. coli R1 plasmid[99,128–130]. Although a structure has yet to be reported for aplasmid type I Walker-type motor protein, the structure of thechromosomal Walker-type protein, T. thermophilus Soj, which isrelated to the type Ib motor proteins, has been described [131].Structures of R1 ParM and T. thermophilus Soj combined withEM studies on several type II and I motor proteins have revealedimportant insight into how these proteins mediate separation ofreplicated plasmids.

Structures of the T. thermophilus Soj protein

The T. thermophilus Soj protein collaborates with the T. thermo-philus Spo0J protein to play an important, yet incompletelycharacterized, role in chromosome segregation. Soj is alsorequired together with Spo0J for stable maintenance of a plasmidbearing the parS centromere site, suggesting that they mediateplasmid segregation [69]. Localization studies of Soj have re-vealed dynamic oscillations of the protein similarly to oscillationsobserved for the type Ib Walker-type protein pB171 ParA[101,102,132]. Soj has also been shown to form nucleoprotein fila-ments, but in a DNA-dependent manner [131]. The structureof T. thermophilus Soj was solved in its apo and ADP-boundforms. In addition, the structure of the ATPase hydrolysis-deficient mutant, Soj(D44A), was solved bound to ATP [131].



Figure 7 Structure of T. thermophilus Soj

On the left (red) is the structure of apo Soj. On the right is the structure of Soj(D44A), whichforms a nucleotide sandwich dimer (one subunit is red and the other green) upon binding ATP.The ATP molecules are shown as CPK (Corey–Pauling–Koltun) with carbon, nitrogen, oxygenand phosphorous atoms coloured cyan, blue, red and magenta respectively [131]. Modelledusing PyMOL (DeLano Scientific).

The structure confirmed previous predictions, that the proteincontains a canonical Walker-type fold (Figure 7).

The Soj structure shows the highest homology with the celldivision regulator MinD [133]. Accordingly, Soj consists of acore of twisted β-strands surrounded by α-helices. The α-helicescluster on either side of the β-sheet formed by one antiparallel β-strand and seven parallel β-strands. The Soj apo and ADP-boundforms are monomers, whereas ATP binding to Soj(D44A) leadsto formation of an ATP ‘nucleotide sandwich dimer’. Notably,the formation of such a dimer had been predicted previously, butnot observed until the structure of the Soj(D44A)–ATP complex[131]. Comparison of apo and ADP-bound Soj, reveals that ADPbinding is accompanied by minor structural changes in the P-loop region wherein residues GGVG (Gly-Gly-Val-Gly) becomeless extended as they make multiple interactions with the ADPphosphate moieties. Upon ATP binding, dimerization is inducedby numerous hydrogen bonds between residues on adjacentsubunits (Figure 7). However, the key interaction in mediatingthe formation of dimer is provided by the Walker box signaturelysine residue, Lys15, which contacts the α- and γ -phosphates ofthe ATP bound in the other subunit.

The formation of Soj polymers requires not only ATP, but alsonon-specific DNA as well [131,134]. A recent study aimed atelucidating what role Soj DNA binding might play in segregationidentified several surface arginine residues, conserved amongchromosomal Soj proteins, that were essential for DNA binding,but appeared to have no significant role in nucleotide binding[134]. Remarkably, creation of a DNA-binding-deficient Sojprotein by mutation of these residues led to a partition-deficientphenotype in a model plasmid partitioning system in E. coli. Thisfinding clearly demonstrated that DNA binding by Soj is essentialfor its segregation function and also suggests a connectionbetween the DNA-binding and -polymerization functions of Soj insegregation [134]. However, the arginine residues that are criticalfor DNA binding by Soj proteins are not conserved among thetype Ib ParA proteins. Moreover, previous studies on the type IbpB171 ParA and ParF motor proteins and the type Ia SopA proteinindicated that DNA binding is not required for polymer formationby these proteins. Rather, ATP binding seems sufficient to inducethe formation of polymers by the type Ib and Ia motor proteinsand the addition of the requisite centromere-binding protein–cen-tromere complex stimulates the process [34,102].

Structures and EM studies on the R1 ParM protein

Before high-resolution structures were available, the type II ParMprotein had been predicted to belong to the actin superfamily.

Figure 8 Structures of actin-like ParA homologue, E. coli R1 ParM

(A) Crystal structure of R1 ParM in its apo state [129]. (B) Structure of the R1 ParM ADP-boundstate. Comparison with the apo structure reveals that ADP binding induces a 25◦ relative closureof the two domains [129]. (C) Structure of eukaryotic actin bound to ADP [143]. In all structures,helices are coloured cyan, β-strands are coloured magenta and loop regions are colouredlight pink. Also labelled are subdomains Ia, Ib, IIa and IIb. Modelled using PyMOL (DeLanoScientific).

Other bacterial proteins that are actin superfamily membersinclude DnaK, FtsA and MreB [135,136]. Crystal structures ofParM in its apo and ADP-bound form confirmed that it hasan actin-like fold and, accordingly, consists of a two-domainarchitecture [129]. These two domains, called I and II, aredelineated further into Ia and Ib and IIa and IIb (Figure 8). Thelarger subdomains, Ia and IIa, share a common fold consisting ofa five-stranded β-sheet surrounded by three α-helices. In contrast,the two smaller subdomains are much more diverse structurally.Comparison of the apo ParM and ParM–ADP structures revealedthat domains I and II act as rigid bodies and undergo a 25◦ rotationupon binding ADP [129] (Figures 8A and 8B). Interestingly,analysis of the ADP-binding pocket of ParM revealed nospecificity-determining interactions between ParM residues andthe adenine base. Indeed, a recent study showed that, in additionto ATP, ParM can bind GTP. In this study, the structure of the R1ParM was solved bound to GDP and the R1 ParM–p[NH]ppG(guanosine 5′-[β,γ -imido]triphosphate) complex structure wasobtained by soaking p[NH]ppG into ParM–GDP crystals [99].The ParM–ADP, ParM–GDP and ParM–p[NH]ppG structures areessentially identical in their conformations, which suggests thatnucleotide binding stabilizes the closed conformation.

Although actin is a well-known filament-forming protein,several actin family members have roles unrelated to filamentor polymer formation. For example, DnaK functions in proteinfolding, and FtsA is involved in the regulation of cell division[137–139]. Thus sequence homology between ParM and actin didnot necessarily dictate filament formation as a mechanismfor ParM action. To address the function of ParM, numerouscellular, biochemical and EM analyses have been carried out[42–45,128,129]. More recent studies by other groups haveprovided additional insights into the structure–function dynamicsof ParM. These studies have firmly established that not only doesParM form filaments, but also that these filaments functiondirectly in plasmid segregation [99,140–141]. Initial hints thatParM forms filaments were obtained from immunofluorescencemicroscopy studies, which revealed that ParM forms highlydynamic polymeric structures that extended along the E. colicell. In addition, ParM was shown to self-assemble in vitro whenATP is present [129]. EM studies on these assemblies of R1ParM filaments revealed the presence of ordered filaments thatwere initially described as actin-like [129]. More recently, twolow-resolution models of ParM filaments have been constructedbased on EM data [99,130]. Specifically, Orlova et al. [130] usedthe high-resolution crystal structures of R1 ParM to carry outimage reconstructions of cryo-EM data of R1 ParM filaments.


14 M. A. Schumacher

Figure 9 EM reconstructions of R1 ParM filaments and type II insertionalpolymerization model

Top: comparison of EM reconstructions from Orlova et al. [130] (left) and Popp et al. [99](right). Left: reprinted by permission from Macmillan Publishers Ltd: Nature Structural andMolecular Biology. Orlova, A., Garner, E., Galkin, V. E., Heuser, J., Mullins, R. D. and Egelman,E. H. (2007) The structure of bacterial ParM filaments. Nat. Struct. Mol. Biol. 14, 921–922.Copyright 2007. http://www.nature.com/nsmb/. Right: reprinted by permission from MacmillanPublishers Ltd: EMBO Journal. Popp, D., Narita, A., Oda, T., Fujisawa, T., Matsuo, H., Nitanai, Y.,Iwasa, M., Maeda, K., Onishi, H. and Maeda, Y. (2008) Molecular structure of the ParMpolymer and the mechanism leading to its nucleotide-driven dynamic instability. EMBO J. 27,570–579. Copyright 2008. http://www.nature.com/emboj/ ParM molecules fitted into the modelare numbered in the Orlova model. Bottom: model of R1 plasmid partition (from Campbelland Mullins [141]). Reproduced from The Journal of Cell Biology, 2007, 179: 1059–1066.Copyright 2007 The Rockefeller University Press. The proposed steps in the model are numbered1–5. Step 1 involves nucleation of filaments. After nucleation, one end of the filaments attachesto one partition complex, and the other end of the filament searches for the partition complexlocated on another plasmid. In step 2, the plasmids diffuse in the cell until they encounter asecond plasmid. Once in close proximity, in step 3, both ends of the filaments are bound bya plasmid, preventing catastrophic collapse. In step 4, because the filaments are now stable,they continue to grow and push the plasmids to opposite cell poles. In step 5, the plasmids arefinally pushed into the cell poles, the force of which destabilizes the interactions of the ParMfilaments with the plasmid. The plasmid is then released, at which point the free end of thefilament is destabilized and rapidly depolymerizes.

Subsequently, Popp et al. [99] used EM, TIRF (total internalreflection fluorescence) microscopy, high-pressure SAXS (small-angle X-ray scattering), and X-ray fibre diffraction to constructthree-dimensional images of ParM–p[NH]ppG filaments andobtain insights into filament dynamics [99] (Figure 9). Theresulting models from these two studies agree that ParM filamentsare left-handed rather than right-handed like F-actin. However, thetwo low-resolution ParM filament models differ quite markedly indetail [99,130]. For example, in the model by Orlova et al. [130],

the ParM molecules within the filaments adopt a conformationthat is even more open than the apo ParM conformation. Incontrast, in the filament model proposed by Popp et al. [99],ParM adopts a closed conformation like that found in the crystalstructures of R1 ParM with bound nucleotides. Despite thesedifferences in detail, both ParM filament models are similar inoverall shape and dimension and thus both studies support theinsertional polymerization model whereby ParM filaments insertbetween plasmids to propel them towards opposite cell poles(Figure 9).

The insertional polymerization model has also recentlyreceived strong support from both in vitro reconstitutionassays and in vivo time-lapse fluorescence microscopy analyses[46,140,141]. Moreover, these studies have provided additionalinsight into this process. The in vivo studies, which followedR1 plasmid dynamics in real time, revealed that ParM filamentsare short-lived and are stabilized only when each end of thefilament is bound to a ParR–parC-associated plasmid [141]. Therates of in vivo ParM polymerization were found to be similarto the timing required for plasmid segregation [46]. The factthat, in vitro, purified ParR and ParM proteins and parC-labelledmagnetic beads alone could drive bead separation and the strongsimilarities between the in vitro and in vivo filament-mediatedseparation of plasmids and parC-labelled beads also stronglysupports the idea that host factors are not required for type IIplasmid segregation. The in vitro reconstitution study also demon-strated the important finding that the filament grows by insertionof additional ParM–ATP molecules at the ParR–parC interface.This insertional polymerization causes the ParM filaments toextend bidirectionally, moving the two plasmids to oppositecell poles. The in vivo studies suggest that, after reaching thepoles, the force of colliding into the ends of the cell lead todissociation of the filament ends from ParR–parC, promptingthe rapid depolymerization of the filament [141] (Figure 9).Indeed, it has been demonstrated that pressure applied to afilamentous structure such as actin can lead to loss of non-covalent interactions, bound nucleotides and susceptibility todepolymerization [142]. This knowledge provides additionalinsight into how the filaments depolymerize at the correct timeafter plasmid separation. Specifically, ParR is known to stimulatethe ATPase activity of ParM. Because ParR is in contact withParM, it will hasten the formation of ParM–ADP (or ParM–GDP)within the filament. However, as long as ParR is bound to the endsof the filaments, it strongly stabilizes the polymer form of ParM.But, after separating, the plasmids will be forced against the cellpoles [141]. This force will dissociate the ends of one or bothParM filaments from its interaction with the partition complex onthe plasmid. The end subunit of ParM, which had previously beenin contact with ParR, will probably exist in the NDP-bound state.Release of the protective partition complex contacts will allowrelease of the NDP molecule. According to the recent ParM–GDP and ParM–p[NH]ppG structures, the NTP-bound and NDP-bound states of ParM are in the same conformation, which isdistinct from the apo conformation [99]. Thus release of the NDPand subsequent conversion into the apo form will lead to domainrotation, which in turn will cause the loss of one subunit from theend, stimulating the disruption of the entire filament.

One problem with the insertional polymerization model is thatit necessitates that each end of the ParM filament interacts withan identical ParR–DNA partition complex. However, structuralinformation indicates that ParM filaments exhibit polarity,meaning they have a plus end and a minus end. To examinehow the different ends of the ParM filament may interact equallywith an identical partition complex, we docked the ends of the R1ParM filament model obtained from EM on to the pSK41 partition



Figure 10 Molecular model of type II segregation

(A) Model of pSK41 segrosome assembly. To construct this model, the pSK41 partition complex structure and the Orlova (for which co-ordinates were deposited) ParM filament model were used[90,130]. As can be seen from this model, the ParM filament can be embraced within the partition complex pore without steric clash and can favourably interact with the C-terminal flexible domainsof the ParR molecules, found at high concentrations within the pore. (B) In this pSK41 segregation model, the ParM filament is captured between two partition complexes on different plasmids.The filament and partition complexes are drawn roughly to scale. Centromere DNA is coloured brown, the ParR molecules are magenta, green, blue and yellow, and ParR molecules participating inspreading are cyan. ParR flexible C-domains, which interact with the ParM filament, are represented as half circles. The ParM filament is represented as a yellow surface rendering. Modelled usingPyMOL (DeLano Scientific).

complex superstructure [90,130]. Remarkably, this modellingrevealed that the filament can readily fit within the partitioncomplex pore (Figure 10). Further modelling shows that this istrue even when accounting for the addition of the ParR C-terminaldomains, which were not present in the structure [90]. As canbeen seen in Figure 10(A), ParM filament polarity would not be aproblem because each end of the filament would encounter ParRmolecules in multiple orientations (Figure 10). Furthermore, theC-terminal region of ParR, which interacts with ParM, is highlyflexible, thereby permitting it to dock on to ParM moleculesthat are arranged in any orientation. An additional feature of thepartition complex structure that is likely to be important for itsinteraction with ParM filaments is that its pore region displays avery high local concentration of ParR C-terminal tails which areperfectly poised for ParM filament interaction and capture [90].ParR spreading could help further encase the ParM molecule andincrease the likelihood of productive ParM binding.

Could this proposed partition complex capture mechanismapply to other par systems?As noted, although ParR/ParB proteinsshow little sequence identity, recent structures of the type Iband type II centromere-binding proteins have revealed a strikinghomology in that both contain RHH DNA-binding domains. Infact, the RHH fold is an excellent partition complex-formingmotif because binding to tandem centromere sites would promoteits co-operative polymerization and the formation of higher-order superstructures. The combined structural data on type IIParR proteins and partition complexes, including EM data onthe R1 partition complex, the crystal structure of pB171 ParRand the crystal structure of the pSK41 partition complex suggestthat type II partition complexes form highly similar superhelicalstructures. It might be anticipated that type Ib centromere-binding proteins, which also contain RHH motifs, form relatedsuperstructures. We speculate that the specific conformation ofeach of the partition complex and segrosomal superstructures islikely to be dictated by the polymer structure of the motor proteinbound by each partition complex.

CONCLUSIONS

DNA segregation is an essential process for the survival of allspecies. The segregation of plasmid DNA requires only three

components and thus is an ideal model system to study the detailedmechanisms involved in this process at the atomic level. Thestructures of bacterial chromosomal and plasmid partition proteinsand their DNA complexes that have been obtained over the last5 years have shed significant light on the molecular mechanismsof segregation of several types of partition systems. In fact,studies on the type II system have revealed that three components,the centromere-binding protein, ATPase and centromere site,can orchestrate DNA separation without the addition of cellularfactors. It has also been established unequivocally that ParM formsfilaments in an ATP- or GTP-dependent manner, and formationof these filaments drives DNA partition. Stabilization of thesefilaments requires interaction of filament ends with the partitioncomplex superstructure, which is formed by the binding of ParRmolecules to the centomere. The first high-resolution structureof a higher-order partition complex, that of the type II pSK41partition complex, revealed that the ParR–centromere complex isa superhelical structure with dimensions suitable for capture ofthe ParM filaments [90]. The position of the ParM-binding siteswithin the flexible C-terminal region of ParR indicates how it canbind the dissimilar ends of the ParM filament.

Despite the substantial progress in delineating a molecular un-derstanding of type II plasmid partition, many questions remain.For example, how do ParR and other centromere-binding proteinsmediate the seemingly opposing functions of ATPase stimulationand polymer stabilization? Structural studies on centromere-bind-ing proteins bound to their requisite motor proteins should helpclarify this paradox. Although many of the steps of type IIpartition are understood at the molecular level, much less isknown about type I segregation. Unexpected structural homologyrevealed between the type II and type Ib centromere-bindingproteins suggests that the type Ib centromere-binding proteinsmay form similar superstructures when interacting with theircentromere sites; however, this remains to be demonstrated. Also,the types of partition complexes formed by the type Ia proteinsare, at this time, completely unknown. The presence of conservedHTH and motif B domains hints at a mechanism involvingDNA spreading as mediated by the combination of these motifs.Perhaps the most unresolved issue regarding type I partition isthe molecular mechanism(s) utilized by type I motor proteins inDNA separation. In fact, no structures are available for a plasmidtype I motor protein and thus how they may form polymers and


16 M. A. Schumacher

bind nucleotides remains a mystery. Mounting evidence suggeststhat polymer formation by type I motor proteins is importantin segregation. If this is the case, do these proteins utilize aninsertional polymerization mechanism similar to that of type II parsystems? Support for a common mode of polymer-driven plasmidseparation comes from recent findings that putative type III andIV par systems employ a tubulin-like GTPase and a coiled-coilprotein respectively for partition. Thus the future challenges areto fill in the missing pieces of the puzzle left in the type II partitionreaction and to determine the molecular mechanism(s) used by thetype Ia and Ib systems, as well as newly identified par systems, toform partition complexes and drive plasmid separation. Structuralstudies combined with biochemical and cellular analyses will beneeded to address these issues and, hopefully, in so doing, provideatomic-level snapshots of each step in the partition reaction.

I acknowledge support from the Burroughs Wellcome (Burroughs Wellcome CareerDevelopment Award 992863), the University of Texas M. D. Anderson Cancer CenterTrust Fellowship and the National Institutes of Health. I thank Professor Finbarr Hayes forhelpful discussions and comments. I apologize to those whose work was not discussed.

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Received 15 February 2008/18 March 2008; accepted 18 March 2008Published on the Internet 25 April 2008, doi:10.1042/BJ20080359


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