Plasmid 53 (2005) 126–136

www.elsevier.com/locate/yplas

Review

Plasmid rolling-circle replication: highlights of two decadesof research

Saleem A. Khan*

Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

Received 20 December 2004Available online 22 January 2005

Communicated by Dhruba K. Chattoraj

Abstract

This review provides a historical perspective of the major findings that contributed to our current understanding ofplasmid rolling-circle (RC) replication. Rolling-circle-replicating (RCR) plasmids were discovered approximately 20years ago. The first of the RCR plasmids to be identified were native to Gram-positive bacteria, but later such plasmidswere also identified in Gram-negative bacteria and in archaea. Further studies revealed mechanistic similarities in thereplication of RCR plasmids and the single-stranded DNA bacteriophages of Escherichia coli, although there wereimportant differences as well. Three important elements, a gene encoding the initiator protein, the double strand origin,and the single strand origin, are contained in all RCR plasmids. The initiator proteins typically contain a domaininvolved in their sequence-specific binding to the double strand origin and a domain that nicks within the double strandorigin and generates the primer for DNA replication. The double strand origins include the start-site of leading strandsynthesis and contain sequences that are bound and nicked by the initiator proteins. The single strand origins arerequired for synthesis of the lagging strand of RCR plasmids. The single strand origins are non-coding regions thatare strand-specific, and contain extensive secondary structures. This minireview will highlight the major findings inthe study of plasmid RC replication over the past twenty years. Regulation of replication of RCR plasmids will notbe included since it is the subject of another review.� 2005 Elsevier Inc. All rights reserved.

Keywords: Plasmid rolling-circle replication; Initiator proteins; Double strand origins; Single strand origins; DNA helicase

0147-619X/$ - see front matter � 2005 Elsevier Inc. All rights reserv

doi:10.1016/j.plasmid.2004.12.008

* Fax: +1 412 624 1401.E-mail address: Khan@pitt.edu.

1. Introduction

Rolling-circle-replicating (RCR) plasmids areubiquitous in Gram-positive bacteria (for detailed

ed.

S.A. Khan / Plasmid 53 (2005) 126–136 127

reviews, see del Solar et al., 1993a, 1998; Khan,1997, 2000, 2004). These plasmids appear to haveevolved from a few common ancestors. So far,more than 200 RCR plasmids have been identifiedand these can be divided into more than a dozenfamilies (http://www.essex.ac.uk/bs/staff/osborn/DPR_home.htm) based on sequence homologiesin their initiator (Rep) proteins and the doublestrand origin of replication (dso). Plasmids ofmany RCR families also share homology in theirrep genes and dso with the single-stranded (ss)DNA bacteriophages of Escherichia coli. AlthoughRCR plasmids are abundant in different Gram-po-sitive bacteria, they have also been reported inmany Gram-negative organisms and in archaea.RCR plasmids are usually less than 10 kb in size,have multiple copies and are tightly organized.Many RCR plasmids carry antibiotic and heavymetal resistance genes, while others are cryptic.Some RCR plasmids also carry genes for mobiliza-tion and transfer, and genes encoding site-specificrecombinases. Furthermore, many RCR plasmidscontain insertion sequences and transposons. Bothbroad and narrow host range RCR plasmids havebeen described and there is evidence for their hor-izontal transfer. All RCR plasmids encode initia-tor proteins that have (or presumed to have)origin-specific binding and nicking–closing activi-ties. The dsos of RCR plasmids contain both thebinding (bind) and nicking (nick) sites for the

Fig. 1. A model for plasmid RC re

Rep proteins. The nick site is highly conserved inthe dsos of all plasmids belonging to a particularfamily, while the Rep binding sites are less well-conserved. Thus, the specificity of the Rep proteinin replication is determined by its specific interac-tion with its cognate dso. Initiation of replicationrequires both the Rep binding and nicking sites,while termination of plasmid RC replication canbe promoted by a smaller sequence containingonly the nick site. The single strand origin (sso)of RCR plasmids is important for the conversionof the displaced leading strand to the double-stranded (ds) form. Unlike the dsos, the ssos arenot necessarily conserved in RCR plasmidsbelonging to the same family. The role of the initi-ator proteins, dsos, ssos and host proteins in plas-mid RC replication is highlighted below.

2. A model for plasmid rolling-circle replication

Our current understanding of the mechanism ofplasmid RC replication can be summarized as fol-lows based on studies with the plasmids of thepT181 family (Fig. 1). The Rep protein interactswith its specific bind sequence located within theplasmid dso. The Rep–dso interaction may resultin a sharp bend in the DNA and/or generationof a hairpin in which the Rep nick site is locatedin the single-stranded loop. The Rep protein then

plication. See text for details.

128 S.A. Khan / Plasmid 53 (2005) 126–136

nicks the dso at the nick sequence and becomescovalently attached to the 5 0 phosphate througha tyrosine residue present in its active site. Repproteins also recruit a DNA helicase (PcrA in thecase of Gram-positive bacteria) through a specificprotein–protein interaction. The helicase then un-winds the DNA and the single-stranded DNAbinding (SSB) protein coats the displaced ssDNA.Leading strand replication initiates by extensionsynthesis at the free 3 0 OH end at the nick siteby DNA polymerase III and proceeds until theleading strand has been fully displaced. Once thereplication fork reaches the termination site, i.e.,the regenerated dso, DNA synthesis proceeds toapproximately 10 nucleotides (nt) beyond theRep nick site (Fig. 1). The second, free monomerof the Rep protein then cleaves the displacedssDNA. Following a series of cleavage/rejoiningevents, the circular, leading strand is releasedalong with a relaxed, closed circular DNA contain-ing the newly synthesized leading strand. ThedsDNA is then supercoiled by the host DNA gyr-ase. The Rep protein in which the active tyrosineof one monomer is covalently attached to the 10-mer oligonucleotide is released (Fig. 1). This formof Rep, termed RepC/RepC* for pT181, whichhas catalyzed one round of leading strand replica-tion is inactive in further replication. The ssDNAreleased after leading strand synthesis has beencompleted is converted to dsDNA utilizing thesso and host proteins. This involves synthesis ofan RNA primer by the RNA polymerase followedby extension of the primer by DNA polymerase Iand subsequent DNA synthesis by DNA polymer-ase III. Finally, the DNA ends are joined by DNAligase and the resultant dsDNA is converted to thesupercoiled (SC) form by DNA gyrase.

3. The discovery and early studies on RCR plasmids

The initial evidence that there was somethingunusual about replication of small plasmids inStaphylococcus aureus came from studies on thein vitro replication of the pT181 plasmid (Khanet al., 1981). These studies showed that replicationof pT181 in staphylococcal cell-free extracts wasnot significantly affected in the absence of rNTPs

and appeared not to require RNA primers for plas-mid replication. The first direct evidence in supportof plasmid RC replication was provided by thedemonstration that the purified initiator proteinencoded by plasmid pT181 had origin-specific nick-ing–closing activity (Koepsel et al., 1985a,b). Fur-thermore, the protein was shown to be covalentlyattached to the 5 0 phosphate at the nick site and afree 3 0 OH end was exposed at the nick site (Koep-sel et al., 1985b). Subsequent studies showed thatpT181 replication initiated by covalent extensionof the DNA strand nicked by the RepC initiatorprotein (Koepsel et al., 1986). These results sug-gested that pT181 replicates by a RC mechanism.The sequence at the initiator nick site also showedhomology to the sequence at the origin of replica-tion of the ssDNA phage f1 of E. coli (Baas,1985; Koepsel et al., 1985b). This suggested amechanistic similarity between the replication ofplasmid pT181 and the ssDNA bacteriophages ofE. coli (Baas, 1985). In vivo support for plasmidRC replication came from studies demonstratingthat plasmid pC194 of S. aureus existed in bothds and ss forms in Bacillus subtilis and S. aureus.(te Riele et al., 1986a,b). Furthermore, the ssDNAwas circular and corresponded to only one of thetwo strands of the pC194 DNA (te Riele et al.,1986a,b). Subsequently, utilizing plasmids contain-ing two tandem copies of the dso, it was convinc-ingly shown both in vivo and vitro that plasmidspC194 and pT181 of S. aureus replicate by an RCmechanism and that ssDNA was an intermediatein plasmid replication (Gros et al., 1987; Iordane-scu and Projan, 1988; Murray et al., 1989). Furtherstudies in several laboratories identified numerousplasmids in Gram-positive bacteria that were eithershown to or predicted to replicate by an RC mech-anism (Andrup et al., 2003; Meijer et al., 1998;Seery et al., 1993). Observations soon followed thatestablished that RCR plasmids were not exclu-sively confined to Gram-positive bacteria andRCR plasmids such as pKYM were identified inGram-negative organisms and plasmid pGT5 in ar-chaea (Marsin and Forterre, 1999; Yasukawa et al.,1991). Recent studies have identified many addi-tional RCR plasmids in a variety of Gram-negativebacteria (http://www.essex.ac.uk/bs/staff/osborn/DPR_home.htm).

S.A. Khan / Plasmid 53 (2005) 126–136 129

4. Initiator proteins of RCR plasmids

Major advances in our understanding of themolecular events during the initiation and termina-tion of plasmid RC replication were provided bythe characterization of the biochemical activitiesof the initiator proteins (Koepsel et al., 1985b,1986; Moscoso et al., 1995a; Thomas et al., 1990,1995). RCR initiator proteins bind to their cognatedsos with high affinity in a sequence-specific man-ner and are critical for the assembly of the replica-tion initiation complex. These initiators alsogenerate a site-specific nick within the dso thatserves as the primer for the initiation of DNA rep-lication (Koepsel et al., 1985b; Moscoso et al.,1995b; Thomas et al., 1988). Since the nick se-quence is highly conserved in all members of a par-ticular plasmid family, the sequence-specificinteraction between Rep and dso provides thespecificity in plasmid replication (Dempsey et al.,1992a; Iordanescu, 1989; Moscoso et al., 1995b;Wang et al., 1992; Zock et al., 1990). Structure–function analyses of the Rep proteins of thepT181 family have shown that they contain a se-quence-specific DNA binding domain and a nick-ing domain (Dempsey et al., 1992a,b; Wanget al., 1992; Fig. 2). A tyrosine residue is involvedin nicking at the origin, and for several plasmids ithas been shown that the initiator becomes cova-lently attached to the 5 0 end of the DNA througha phosphotyrosine linkage (Dempsey et al., 1992b;Thomas et al., 1988). The initiator proteinsbelonging to a particular family have highly con-served nicking domains (for reviews, see del Solar

Fig. 2. Domain structure of the Rep proteins of the pT181 family. AmpT181. The DNA binding and nicking domains of the proteins are shodso) and the carboxyl terminal region containing the sequence-specificthe rest of the protein.

et al., 1998; Ilyina and Koonin, 1992; Khan, 1997;Projan and Novick, 1988). On the other hand, theyare quite divergent in their sequence-specific DNAbinding domains (Projan and Novick, 1988). Thisexplains why all the initiators of a particular fam-ily can nick–close the DNA of all such plasmids,but are highly specific for supporting replicationfrom their cognate dsos. However, the initiatorscan support limited replication of heterologousplasmids of the same family in the absence of theircognate origins (Iordanescu, 1989; Wang et al.,1992; Zock et al., 1990).

Specific amino acids involved in sequence-spe-cific interaction between Rep and dsos of thepT181 family members have been identified andit has been shown that switching six amino acidsbetween such proteins can switch their replicationspecificity (Dempsey et al., 1992a,b; Wang et al.,1992). Mutational analysis of the RepC proteinof plasmid pT181 showed that it is nicking–closingand non-covalent DNA binding activities can beuncoupled and both of these activities are essentialfor replication (Dempsey et al., 1992b). The initia-tor proteins of the plasmids of the pT181 familyact as dimers during replication (Chang et al.,2000; Rasooly and Novick, 1993; Rasooly et al.,1994). Utilizing the RepC protein of plasmidpT181 containing various combination of wild-type, bind� or nick� monomers, it was shown thatone monomer of the dimeric initiator is sufficientfor sequence-specific dso binding and nicking(Chang et al., 2000). Furthermore, the monomerthat promotes sequence-specific binding to thedso was also found to nick the DNA to initiate

ino acid numbering corresponds to that of the RepC protein ofwn. The amino terminal end of the Rep proteins (encoded by theDNA binding domain are much less conserved as compared to

130 S.A. Khan / Plasmid 53 (2005) 126–136

replication (Chang et al., 2000). Interestingly,whereas Tyr-191 of the pT181 initiator was re-quired for nicking at the origin to initiate replica-tion, it was dispensable for termination,suggesting that alternate amino acids in the initia-tor may promote termination but not initiation(Chang et al., 2000).

The RepA protein of plasmid pC194 was shownto act as a monomer and its Tyr-214 residue wasfound to nick the DNA during the initiation ofreplication and become covalently attached tothe DNA (Noirot-Gros et al., 1994). However, aglutamate residue located in close proximity toTyr-214 cleaves the DNA during the terminationevent (Noirot-Gros et al., 1994). This results inthe release of the initiator after supporting oneround of plasmid replication. Interestingly,replacement of the above glutamate residue witha tyrosine promotes reinitiation of replication afterthe termination of one round of replication(Noirot-Gros and Ehrlich, 1996). This process issimilar to the activity of the gene A protein of /X174 (Van Mansfeld et al., 1986), and suggeststhat the plasmid initiators have evolved such thatthey support only one round of replication whichis important for regulation of their replication.The initiators of the plasmid pT181 family alsosupport only one round of plasmid replication.This is due to their inactivation during the termi-nation step by the attachment of an approximately10 nucleotide long sequence present downstreamof the Rep nick site to their active tyrosine residue(Rasooly and Novick, 1993). The Tyr-99 residue ofthe RepB protein of the pMV158 plasmid wasshown to be involved in origin nicking, but RepBhas not been shown to be covalently attached tothe DNA after nicking (Moscoso et al., 1997).However, it is likely that there is transient attach-ment of the RepB protein to the DNA. No struc-tural information is currently available on theinitiator proteins encoded by the RCR plasmids.However, recently the three-dimensional solutionstructure of the catalytic domain of the initiatorprotein of the tomato leaf curl virus that replicatesby an RC mechanism has been solved (Campos-Olivas et al., 2002). This initiator protein sharesseveral conserved motifs with the initiators of thepC194 family (Ilyina and Koonin, 1992). This

information is likely to facilitate our understand-ing of the structural features and functions of theinitiator proteins of RCR plasmids.

5. The double strand origins of RCR plasmids and

leading strand replication

The double strand origins of several RCR plas-mids were identified by both in vivo and in vitrostudies. The dsos are generally less than 100 bp insize (for reviews, see del Solar et al., 1998; Khan,1997). The dsos of RCR plasmids contain boththe bind and nick sequences for the Rep proteins.The nick site is highly conserved in the dsos ofall plasmids belonging to a particular family, whilethe Rep binding sites are less well-conserved (Fig.3). The dsos of many RCR plasmids were found tocontain structural features such as cruciforms andhairpins (Fig. 3, Gros et al., 1987; Moscoso et al.,1995a; Noirot et al., 1990; Wang et al., 1993), andstatic and initiator protein-enhanced bending ofthe dsos of the pT181 and pMV158 family mem-bers has also been reported (Koepsel and Khan,1986; Perez-Martin et al., 1988). These structuralfeatures are likely to be involved in efficientrecruitment/utilization of the initiator protein tothe origin and the initiation of replication. In someRCR plasmid families, the dsos were found to belocated adjacent to the rep genes, while in othersthey were present within the sequence encodingthe initiator proteins (del Solar et al., 1998; Khan,1997). Plasmids containing two copies of the dso ina direct orientation were used to identify the se-quence requirements for the initiation and termi-nation of replication of plasmids of the pT181and pC194 families (Gennaro et al., 1989; Groset al., 1987, 1989; Iordanescu and Projan, 1988;Murray et al., 1989). While the signals for initia-tion and termination of replication were found tooverlap, a larger region of dso containing boththe initiator bind and nick sequences were requiredfor initiation (Gros et al., 1987; Zhao and Khan,1996).

Termination, on the other hand, was found torequire the nick sequence but the major Rep bind-ing site was dispensable (Iordanescu and Projan,1988; Zhao and Khan, 1997). For example, while

Fig. 3. Sequence alignment of the dsos of the plasmids of the pT181 family. Only the bottom strand of the DNA containing the Repnick site is shown. Replication of pT181 proceeds in a rightward direction. Numbering corresponds to the pT181 sequence. Nucleotidesin pC221 and pS194 which are identical to pT181 are shown in capitals. The initiation region containing the Rep nick site is almostidentical while the specificity region containing the specific initiator binding site (overlined) is less well-conserved. The inverted arrowsrepresent the two arms of the inverted repeat element (IRII) that contains the Rep nick site. The predicted structure of the IRII regionis shown at the bottom. L and R represent the left and right arms of IRII, respectively.

S.A. Khan / Plasmid 53 (2005) 126–136 131

a 70 bp region of the dso was required for optimalinitiation of plasmid pT181 replication, an inter-nal 24 bp region was sufficient for termination(Gennaro et al., 1989; Zhao and Khan, 1996). Itis likely that high-affinity association of Rep withits binding sequence is important for the recruit-ment of host replication proteins and initiationof replication. However, since the Rep protein isexpected to be in close proximity to the regener-ated dso during the termination step (since it iscovalently attached to the DNA; see Fig. 1), itstransient interaction with the nick sequence maybe sufficient to promote the termination event.Very little is known about the role of host pro-teins in the termination of plasmid RC replica-tion, except that the replication terminationprotein does not appear to be involved in this step(Kaul et al., 1994).

6. The single strand origin and lagging strand

synthesis of RCR plasmids

The parental leading strand of plasmid DNAthat is displaced upon synthesis of the new DNAstrand by the RC mechanism is converted to theds form utilizing the sso. The sso was first identi-fied in plasmid pT181 and shown to contain se-quences that can form a folded structure thatwas important for its function (Gruss et al.,1987). Deletion of the sso caused plasmid instabil-ity, a marked reduction in copy number, and re-sulted in the accumulation of large quantities ofcircular, leading strand DNA (Gruss et al.,1987). Subsequently different classes of ssos,namely ssoA, ssoT, ssoU, and ssoW, were identi-fied in several RCR plasmids based on structuraland/or sequence similarities (Andrup et al., 1994;

132 S.A. Khan / Plasmid 53 (2005) 126–136

Boe et al., 1989; del Solar et al., 1993b; Krameret al., 1995; Madsen et al., 1993; Meijer et al.,1995; Seegers et al., 1995; Zaman et al., 1993).The sso s are in non-coding regions, are strand-specific, and not necessarily conserved in the plas-mids of the same family. The ssos are generallylocated immediately upstream of the dsos such thatthey are not exposed in an ss form until the leadingstrand has been almost fully synthesized (see Fig.1). The ssoA and ssoW type origins were foundto function efficiently only in their native hostswhile the ssoT and ssoU could support laggingstrand synthesis in several different hosts.

An in vitro system was developed for the repli-cation of ssDNAs containing ssoA-type originsand used to demonstrate that DNA synthesis initi-ated within the ssoA sequences and required solelythe host proteins (Birch and Khan, 1992). Both invivo and in vitro studies showed that DNA poly-merase I was required for the ssfi ds synthesis,and both its polymerase and 5 0 fi 3 0 exonucleaseactivities were involved in this function (Diazet al., 1994; Kramer et al., 1997). The ssoA-typeorigins were found to contain two conserved ele-ments, RS-B and the CS-6 sequences (Dempseyet al., 1995; Kramer et al., 1995). In vitro studiesshowed that ssoAs and ssoU contain ssDNA pro-moters and the host RNA polymerase synthesizesapproximately 20 and 45 nt long RNA primers,respectively, that are used for the initiation of lag-ging strand DNA synthesis (Kramer et al., 1997,1998a, 1999). Mutational and electrophoreticmobility-shift analyses (EMSA) showed that theRNA polymerase binds to ssoA sequences, andthe RS-B sequence is important for this binding(Kramer et al., 1997, 1998b). Also, the conservedCS-6 sequence of ssoA s was found to serve asthe termination site for primer RNA synthesis(Kramer et al., 1997, 1998a). Finally, the structuralintegrity of the ssoA was found to be essential forappropriate recognition and primer synthesis bythe RNA polymerase (Kramer et al., 1997,1998b). The ssoU-type origins were also found tobind stably to the RNA polymerase (Krameret al., 1999). It has been reported that while thessoA-type origins are host-specific, the ssoT andssoU origins can support replication in a numberof different Gram-positive bacteria (del Solar

et al., 1993b; Josson et al., 1989; Kramer et al.,1995; Meijer et al., 1995). EMSA studies demon-strated that while the S. aureus RNA polymerasebound with high affinity to the pE194 plasmidwhich is native to S. aureus, it interacted poorlywith the ssoA of the streptococcal plasmidpMV158 (Kramer et al., 1998a). On the otherhand, the ssoU sequence of plasmids pUB110 ofS. aureues and pMV158 of S. pneumoniae boundequally well to both the S. aureus and B. subtilis

RNA polymerases (Kramer et al., 1999). Basedon these results, it was hypothesized that thestrength of RNA polymerase–sso interaction maydetermine the efficiency of lagging strand synthesisin different hosts and may determine, at least inpart, narrow vs. broad host range replication ofRCR plasmids (Kramer et al., 1998a, 1999). Thus,the ability of ssoU-type sequences to function inmany different hosts may be critical for the hori-zontal spread of several RCR plasmids.

7. Role of host proteins in plasmid RCR replication

Limited information is available on the hostproteins that are essential for plasmid RC replica-tion. As discussed above, RNA polymerase andDNA polymerase I are required for plasmid RCreplication. It is also assumed that SSB is requiredfor both leading and lagging strand DNA synthe-sis. The pcrA gene was first identified as being re-quired for plasmid pT181 replication andpredicted to encode a helicase (Iordanescu, 1993;Iordanescu and Basheer, 1991). This gene was alsofound to be essential for cell viability, and pcrA

mutants were isolated that were viable but defec-tive in supporting plasmid pT181 replication (Ior-danescu, 1993; Iordanescu and Basheer, 1991).The pcrA mutants maintained pT181 at a greatlyreduced copy number and accumulated nickedopen circular plasmid DNA. These results, alongwith the homology between PcrA and the UvrDand Rep helicases, suggested that PcrA unwindspT181 DNA during plasmid RC replication (Ior-danescu, 1993; Iordanescu and Basheer, 1991; Pe-tit et al., 1998). The pcrA gene has been identifiedin all the Gram-positive bacteria whose genomeshave so far been sequenced. PcrA was first purified

S.A. Khan / Plasmid 53 (2005) 126–136 133

from Bacillus stearothermophilus and shown tohave 3 0 fi 5 0 helicase activity (Soultanas et al.,1999). Although this PcrA was not very processive,the RepD initiator protein encoded by the pC221plasmid (a member of the pT181 family) increasedits processivity (Soultanas et al., 1999). PcrA actsas a monomer and its crystal structure has beendetermined (Subramanya et al., 1996). The knowl-edge of the three-dimensional structure of the B.

stearothermophilus PcrA should facilitate futurestudies dealing with the role of PcrA-initiator pro-tein interactions in the initiation and terminationof plasmid RC replication.

The PcrA helicase of S. aureus has been purifiedand shown to promote DNA unwinding from theRepC generated nick in the pT181 DNA (Changet al., 2002). A direct physical interaction betweenthe RepC and the S. aureus PcrA helicase was alsodemonstrated (Chang et al., 2002). Furthermore,utilizing cell-free extracts from the pcrA3 mutantthat is defective in pT181 replication, it was shownthat the addition of purified wild-type PcrA re-stored plasmid replication in these extracts (Changet al., 2002). Thus, there is direct biochemical evi-dence that PcrA unwinds the initiator-nickedpT181 DNA and promotes plasmid RC replica-tion. Interestingly, the S. aureus as well as Bacillusanthracis and Bacillus cereus PcrA helicases werefound to have bipolar 5 0 fi 3 0 and 3 0 fi 5 0 helicaseactivities (Anand et al., 2004; Chang et al., 2002;Naqvi et al., 2003). Thus, it is possible that differenthelicase polarities of PcrA may be required for itsrole in plasmid RC replication and its currently un-known function that is essential for cell viability.Plasmids of the pT181 family can be establishedin B. anthracis and B. cereus (Anand et al., 2004).In vitro studies showed that the PcrA helicases ofB. anthracis and B. cereus can substitute for thePcrA helicase of S. aureus during in vitro replica-tion (Anand et al., 2004). Furthermore, a directphysical interaction between RepC and these heli-cases was also demonstrated (Anand et al., 2004).Thus, it is possible that an interaction betweenRCR plasmid initiators and PcrA helicases maybe important in broad host range replication ofRCR plasmids. Although not directly demon-strated, it is likely that PcrA is also required forthe replication of all RCR plasmids in Gram-posi-

tive bacteria. In Gram-negative bacteria which lackPcrA, the UvrD helicase was shown to promote thereplication of RCR plasmids (Bruand and Ehrlich,2000). Earlier studies suggested a requirement forDNA polymerase III in plasmid RC replication(Alonso et al., 1988). Recently two essential DNApolymerases, PolC and DnaE, have been identifiedin Gram-positive bacteria (Dervyn et al., 2001; In-oue et al., 2001). However, it is not yet known ifone or both of these DNA polymerases are re-quired for plasmid RC replication.

8. What questions remain to be answered?

Since the discovery of RCR plasmids two dec-ades ago, major advances have been made in ourunderstanding of the critical events during plasmidRC replication. However, there are several majorgaps in our understanding of the DNA–proteinand protein–protein interactions that are criticalfor this mode of replication. For example, very littleis known about the interaction between PcrA andRep proteins of several RCR plasmid families andthe role, if any, of this interaction in determiningthe host range of RCR plasmids. Further studiesare also necessary for a better understanding ofthe role of ssos in determining the host range ofRCR plasmids. The molecular events involved inthe termination of plasmid RC replication arepoorly understood and should be the subject of fu-ture investigations. For example, whether themovement of the PcrA helicase is blocked duringthe termination step and the role of the initiatorprotein in this process remains to be investigated.The role of host proteins in the termination of plas-mid RC replication is another area that remains tobe explored. Finally, the knowledge of three-dimen-sional structure of the initiator proteins of RCRplasmids in the future should provide important in-sights into the critical roles of these proteins in thereplication and regulation of plasmid replication.

Acknowledgments

I thank members of my laboratory who havecontributed to our work. I also thank members

134 S.A. Khan / Plasmid 53 (2005) 126–136

of the plasmid biology community who have con-tributed greatly to our understanding of the plas-mid rolling-circle replication. The research in theauthor�s laboratory has been supported by GrantsGM31685 and AI55929 from the National Insti-tutes of Health.

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