plasmid rolling-circle replication: recent developments

Molecular Microbiology (2000) 37(3), 477±484

MicroReview

Plasmid rolling-circle replication: recent developments

Saleem A. Khan

Department of Molecular Genetics and Biochemistry,

University of Pittsburgh School of Medicine, Pittsburgh,

PA 15261, USA.

Summary

It is now well established that a large majority of

small, multicopy plasmids of Gram-positive bacteria

use the rolling-circle (RC) mechanism for their repli-

cation. Furthermore, the host range of RC plasmids

now includes Gram-negative organisms as well as

archaea. RC plasmids can be broadly classified into

at least five families, individual members of which are

spread among widely different bacteria. There is sig-

nificant homology in the basic replicons of plasmids

belonging to a particular family, and there is compel-

ling evidence that such plasmids have evolved from

common ancestors. Major advances have recently

been made in our understanding of plasmid RC repli-

cation, including the characterization of the biochemi-

cal activities of the plasmid initiator proteins and their

interaction with the double-strand origin, the domain

structure of the initiator proteins and the molecular

basis for the function of single-strand origins in plas-

mid lagging strand synthesis. Over the past several

years, there has been a `renaissance' in studies on

RC replication as a result of the discovery that many

plasmids replicate by this mechanism, and studies in

the next few years are likely to reveal new and novel

mechanisms used by RC plasmids for their regulated

replication.

Introduction

Plasmids that replicate by a rolling-circle (RC) mechanism

were discovered approximately 15 years ago (Koepsel

et al., 1985; te Riele et al., 1986). These plasmids were

originally thought to be an exception to the rule that most

plasmids replicate by a theta-type mechanism. Although it

was well known that single-stranded DNA (ssDNA) bacterio-

phages of Escherichia coli replicate by a RC mechanism,

the above findings showed that a DNA molecule that is

normally present in a double-stranded (ds) form may also

replicate by a RC mechanism. The initiator (Rep) protein

encoded by plasmid pT181 was originally shown to have

origin binding and nicking-closing activities (Koepsel et al.,

1985). Subsequently, this observation was confirmed with

initiators encoded by several other RC plasmid families as

well (reviewed by Khan, 1997; del Solar et al., 1998). The

various steps involved in the initiation and termination of

plasmid RC replication are summarized in Fig. 1. Repli-

cation initiates when the Rep protein interacts with the

plasmid double-strand origin (dso) through a sequence-

specific interaction. The Rep±dso interaction may result

in a sharp bend in the DNA and the generation of a hairpin

in which the Rep nick site is located in the single-stranded

loop (Koepsel and Khan, 1986; Jin et al., 1996). This is

followed by nicking of the dso by Rep and recruitment of a

DNA helicase and other proteins, such as the single-

stranded DNA-binding protein and DNA polymerase III.

The Rep protein becomes covalently attached to the 5 0

phosphate at the nick site through a tyrosine residue

present in its active site. Leading strand replication initi-

ates by extension synthesis at the free 3 0 OH end at the

nick and proceeds until the leading strand has been fully

displaced. The Rep protein then cleaves the displaced

ssDNA at the regenerated nick site at the junction of the

old and newly synthesized leading strand. After a series of

cleavage/rejoining events, the circular, leading strand is

released along with a relaxed, closed circular DNA con-

taining the newly synthesized leading strand. The DNA is

then supercoiled by DNA gyrase. The ssDNA released

after leading strand synthesis has been completed is con-

verted to the dsDNA form using the single-strand origin

(sso) and the host proteins. It is known that RNA poly-

merase generally synthesizes an RNA primer from the

ssos, and DNA polymerase I extends this primer, followed

by replication by DNA polymerase III. Finally, the DNA

ends are joined by DNA ligase, and the resultant dsDNA

is supercoiled by DNA gyrase. Recent studies have

suggested that ss ! ds replication may play an important

role in narrow- versus broad-host-range replication of RC

plasmids, an observation that provides at least one

important basis for the spread of RC plasmids among

different bacterial species.

Several detailed reviews have been published dealing

Q 2000 Blackwell Science Ltd

Accepted 8 May, 2000. *For correspondence. E-mail [email protected];Tel. (11) 412 648 9025; Fax (11) 412 624 1401.

with plasmid RC replication (Gruss and Ehrlich, 1989;

Novick, 1989; 1998; Khan, 1997; del Solar et al., 1998).

This review will focus on recent developments that clarify

the molecular events that are critical for the initiation and

termination of plasmid RC replication, including Rep±dso

interactions, domain structure of the Rep proteins and the

dso, and a critical analysis of our understanding of the

function of broad- and narrow-host-range ssos.

Leading strand replication

Initiation and termination of replication

The first events during the initiation of RC replication

include a sequence-specific interaction between the dso

and the plasmid Rep protein. The dso contains both the

specific recognition sequence for binding by the Rep

protein as well as its nick site. In some plasmids, such as

those of the pT181 and pC194 families, these sequences

are located adjacent to each other (Koepsel et al., 1986;

Noirot-Gros et al., 1994). However, in many plasmids

belonging to the pE194/pLS1 family, the Rep binding and

nicking sequences are separated by a much longer dis-

tance of approximately 85 nucleotides (del Solar et al.,

1998). The Rep protein binds to its specific recognition

sequence (approximately 30 nucleotides long) within the

dso through its DNA-binding domain. In the case of

plasmids of the pT181 family, this interaction has been

shown to result in structural changes within the dso, such

as DNA bending and cruciform extrusion (Koepsel and

Khan, 1986; Jin et al., 1996). This, in turn, exposes the

nick site in the DNA that is then cleaved by the Rep

protein through its active tyrosine residue. The Rep pro-

teins of the plasmid pT181 family, similar to the gene A

protein of fX174, are covalently attached to the 5 0 end at

the nick through an active tyrosine residue (Brown et al.,

1984; Van Mansfeld et al., 1986; Dempsey et al., 1992a;

Thomas et al., 1995). However, in the case of the Rep

proteins of the pLS1 family, there is only a transient

attachment between the active tyrosine residue and the

DNA (del Solar et al., 1998). An event that may occur

simultaneously (or precede) Rep nicking is the recruit-

ment of a host helicase to the dso. In Gram-positive

bacteria, such as Staphylococcus aureus and Bacillus

subtilis, the host-encoded PcrA helicase has been shown

to be involved in the replication of RC plasmids such as

pT181 and pC194 (Iordanescu, 1993; Petit et al., 1998).

PcrA shares considerable homology with the Rep and

UvrD helicases of Escherichia coli. Interestingly, although

the Rep helicase of E. coli is known to be involved in the

RC replication of ssDNA bacteriophages such as fX174

and M13, plasmid RC replication in E. coli requires the

UvrD helicase (helicase II) (Bruand and Ehrlich, 2000).

Extension synthesis then occurs, facilitated by unwinding

of the helicase, and it is likely that the Rep protein

covalently bound to the 5 0 end of the DNA stimulates DNA

unwinding through its interaction with the helicase

(Soultanas et al., 1999). Replication by DNA Pol III (Alonso

et al., 1988) proceeds until the nick site has been regen-

erated and the parental leading strand fully displaced. At

this stage, it is likely that the movement of the replication

fork is blocked. How does this occur? Very little information

is currently available on this issue. In the case of the RC

replication of fX174, the phage-encoded C protein

appears to inhibit leading strand synthesis and may be

involved in the termination of leading strand synthesis

(Goetz et al., 1988). As RC plasmids encode only a single

positively acting Rep protein, a reasonable possibility is

that this protein interacts with (or is complexed to) the

helicase, and the movement of the replication fork is

blocked through a specific interaction between the Rep

protein and the regenerated dso. Although no termination

(Ter) protein has so far been shown to be involved in the

termination of RC replication, its existence should be investi-

gated. After this event, a series of concerted cleavage/

ligation reactions occurs that results in the displacement

of the parental leading strand DNA and a duplex containing

Fig. 1. A model for the replication of RCplasmids. The Rep protein, as exemplifiedby initiators of the pT181 family, is shownas a dimer, but other Rep proteins may actas monomers or oligomers. See text fordetails.

478 S. A. Khan

Q 2000 Blackwell Science Ltd, Molecular Microbiology, 37, 477±484

the newly synthesized leading strand. The released ssDNA

is then converted to the ds form using a sso.

Domain structure of the Rep proteins

Rep proteins belonging to plasmids of a particular family

have conserved active sites that include the tyrosine

residue involved in nicking-closing at the dso (Khan, 1997).

Thus, a Rep protein can generally nick the dso of all

related plasmids. However, these proteins show specifi-

city in their replication, i.e. they efficiently replicate only

their cognate plasmids, although they may support vari-

able levels of replication of related plasmids in the

absence of a competing, cognate origin (Dempsey et al.,

1992b; Thomas et al., 1995). This specificity of Rep pro-

teins is generally determined by the presence of discrete

DNA-binding domains that are involved in their sequence-

specific, non-covalent interaction with the dso. The domain

structure of the Rep proteins has been extensively studied

for the initiator proteins of the pT181 family and, to a

lesser extent, that of the Rep proteins of the pC194 family

(Dempsey et al., 1992a; Noirot-Gros et al., 1994; Marsin

and Forterre, 1999). The Rep proteins encoded by the

pT181 family members have a modular structure and

contain domains involved in their non-covalent, sequence-

specific binding to the dso as well as domains for origin

nicking-closing (Dempsey et al., 1992a). These two

domains are mutationally separable and, in general, loss

of either the DNA-binding or nicking activity does not sig-

nificantly affect the other activity (Dempsey et al., 1992a).

A region consisting of approximately 50 amino acids

located near their carboxyl-terminal is required for their

DNA-binding activity (Dempsey et al., 1992b). Within this

region, a short amino acid sequence is highly variable

among such proteins. Experiments have shown that a

maximum of six amino acids located in this region are

sufficient to determine the DNA-binding specificity of the

initiator proteins. Exchange of these six amino acids

between the Rep proteins of the pT181 family switches

their DNA binding and replication specificity (Dempsey

et al., 1992b). Similarly, the dsos of the plasmids of the

pT181 family contain a variable region that is specifically

recognized by their cognate Rep proteins (Novick, 1989;

Khan, 1997). The Rep proteins of most other plasmid

families are also known to share considerable homology

(Noirot-Gros et al., 1994; del Solar et al., 1998). Although

no information is currently available on their DNA-binding

domains, it is likely that they contain a modular structure

similar to those of the Rep proteins of the pT181 family.

The Rep proteins encoded by the RC plasmids contain

a separate nicking-closing domain that includes the active

tyrosine residue. The amino acids in this domain, includ-

ing the active tyrosine residue, are highly conserved

among the Rep proteins of individual plasmid families

(Khan, 1997; del Solar et al., 1998). The Rep proteins can

generally nick-close all plasmids of the same family

because of the conservation of the nick sequence in the

dsos (Khan, 1997; del Solar et al., 1998). However, the

replication specificity of RC plasmids is determined

through the non-covalent, sequence-specific binding of

Rep to the dso, and the ability of Rep to nick the dso is not

sufficient for the initiation of replication. This presumably

results from the fact that, before nicking at the dso, a

stable nucleoprotein complex (involving at least the dso,

Rep and a helicase) must assemble in vivo. In the

absence of a stable Rep±dso interaction, a replication

initiation complex is unlikely to assemble, and a transient

nick induced by the heterologous Rep protein would not

result in initiation. In the case of the pT181 family initi-

ators, the nicking and DNA-binding domains are sepa-

rated by approximately 80 amino acids. However, it is

likely that these domains are present in close proximity in

the folded structure of the initiator proteins.

Dimeric versus monomeric RC initiators

The RC initiators must generally contain two active

centres, as cleavage at the dso must occur during both

the initiation and the termination steps. In the case of the

gene A protein of fX174 that acts as a monomer, this is

accomplished by two closely spaced tyrosine residues

(Brown et al., 1984; van Mansfeld et al., 1986). On the

other hand, the Rep proteins of the plasmid pT181 family

exist as dimers, and the active tyrosine residue of each

monomer is involved in replication (Rasooly and Novick,

1993; Thomas et al., 1995; Zhao et al., 1998). The Rep

proteins of the plasmid pC194 family appear to act as

monomers, whereas those of the pE194/pLS1 family may

be present as hexamers (Noirot-Gros et al., 1994; Muller

et al., 1995; del Solar et al., 1998). Recent studies provide

a more detailed understanding of the role of individual

monomers of the dimeric pT181 initiator in replication.

Using purified heterodimers of the RepC protein contain-

ing various combinations of either wild-type, DNA binding-

minus or nicking-minus monomers, it was shown that one

monomer with DNA-binding activity is sufficient to target

an initiator dimer to the dso (Chang et al., 2000).

Furthermore, the monomer that binds to the dso must

also nick at the origin for the initiation of replication.

Interestingly, although the active Tyr-191 residue of the

RepC protein of pT181 is absolutely required for nicking

during initiation, it is dispensable for the termination step

(Chang et al., 2000). Current information suggests that,

although Tyr-191 of the second monomer of RepC is

normally involved in DNA transesterification during termi-

nation, an alternate amino acid can perform this function

in its absence. Whether this reaction can be performed by

another tyrosine or an acidic residue in RepC, as is the

Plasmid rolling-circle replication 479


case for RepA of pC194 (see below), remains to be

determined. The RepC protein of pT181 that acts as a

dimer has been shown to be capable of oligomerizing at

the origin in vitro (Zhao et al., 1998). Whether an addi-

tional dimer of RepC has a role during the initiation and/or

termination step remains to be investigated.

The RepA and RepU proteins encoded by the pC194

and pUB110 plasmids, respectively, appear to act as

monomers, as is the case with the gene A protein of

fX174 (Brown et al., 1984; van Mansfeld et al., 1986;

Noirot-Gros et al., 1994; Muller et al., 1995). Interestingly,

although Tyr-214 of RepA encoded by pC194 is involved

in dso nicking during initiation, a closely spaced acidic

residue (Glu-210) is involved in termination by promoting

cleavage of the phosphodiester bond at the regenerated

dso through hydrolysis (in place of transesterification)

(Noirot-Gros et al., 1994). This event also prevents reinitia-

tion of replication. RepA mutants in which Glu-210 has

been replaced by a tyrosine carry out appropriate termi-

nation. Furthermore, such a mutant can promote reinitia-

tion of plasmid replication in a manner similar to that of the

gene A protein of fX174 (Brown et al., 1984; Noirot-Gros

and Ehrlich, 1996). The above studies show a good deal

of flexibility in the functions of the Rep proteins of the

pT181 and pC194 families, as well as both similarity to

and differences from the initiator proteins of ssDNA

bacteriophages that also replicate by a RC mechanism.

Thus, there are at least two major differences in the

biochemical activities of the initiators encoded by the RC

plasmids and ssDNA bacteriophages. First, the initiators

of bacteriophages can reinitiate replication on the same

template DNA, such that replication of a single molecule

can generate several progeny molecules (Brown et al.,

1984; van Mansfeld et al., 1986), whereas the plasmid

initiators are unable to reinitiate replication. Secondly, the

plasmid initiators have been shown to (or expected to) be

inactivated after supporting one round of replication,

whereas the phage initiators are not subject to inactivation

and can promote several initiation events. Many important

questions about the structure and function of the RC

plasmid initiators remain to be answered. So far, no

information is available on the three-dimensional structure

of these proteins. This information will be critical for an in-

depth understanding of the DNA±protein interactions that

occur during the initiation of plasmid RC replication, as

well as to understand how this interaction is accomplished

in the presence of Rep proteins that act as monomers

versus those that act as dimers or oligomers.

Mechanism of inactivation of the Rep proteins

Unlike the ssDNA bacteriophages, replication of RC plas-

mids is tightly regulated. This is usually accomplished by

mechanisms that regulate the levels of the RC initiator

proteins, which are rate-limiting for replication (reviewed

by Novick, 1989; 1998; Khan, 1997; del Solar et al., 1998).

However, for this regulatory process to be effective, there

must be a specific mechanism to prevent Rep reutilization

to ensure a fixed number of initiation events. Among other

possibilities, this may be accomplished by the degradation

of the Rep protein after its utilization in replication or its

modification such that it is inactive in replication. Further-

more, unless the inactivated Rep has an additional role in

the regulation of plasmid replication and copy number, it

should not interfere with the activity of the unused Rep

protein. How is this accomplished? This issue has been

investigated in detail so far only for the Rep proteins of the

plasmid pT181 family, which act as dimers during repli-

cation. The Tyr-191 residue of one monomer of RepC is

covalently linked to the 5 0 end at the nick site during

initiation and, after displacement synthesis, the replication

fork proceeds approximately 10 nucleotides beyond the

regenerated Rep nick site, i.e. the displaced leading

strand is 10 nucleotides longer. At this stage, Tyr-191 of

the second, free monomer cleaves the displaced ssDNA,

and a series of concerted cleavage±rejoining reactions

leads to the release of a dsDNA and the leading strand

DNA. This event results in the attachment of a 10-

nucleotide-long sequence to Tyr-191 of one Rep mono-

mer (Rasooly and Novick, 1993). This initiator, termed

RepC/RepC* for pT181 and RepD/RepD* for pC221, has

been shown to be inactive in nicking of supercoiled

plasmid DNA and initiation of replication, but retains its

DNA binding-activity (Jin et al., 1996; Zhao et al., 1998).

An issue that arises is whether the inactivated Rep can

compete with the wild-type, unused initiator for dso

binding and therefore may inhibit replication. However,

the inactive Rep does not appear to have a significant role

in the regulation of replication, possibly because it binds to

the origin DNA with a reduced affinity compared with the

wild-type protein (Jin et al., 1996; Zhao et al., 1998). It is

possible that the inactivated initiator undergoes a struc-

tural change that results in this protein having a weaker

DNA binding, DNA bending or cruciform extrusion activ-

ities (Koepsel and Khan, 1986; Jin et al., 1996), in addition

to being essentially inactive in nicking of the supercoiled

DNA. Interestingly, this protein retains ssDNA cleavage

activity, an event that is necessary for its termination

activity, which involves cleavage of the displaced leading

strand of the plasmid DNA (Fig. 1). Future studies,

including those at the structural level, are necessary to

identify the molecular basis for the lack of biological

activity of the inactivated initiator proteins of the pT181

family. The RepU protein encoded by the pUB110 plasmid

that belongs to the pC194 family also appears to be

inactivated by the attachment of an oligonucleotide after

supporting one round of replication (Muller et al., 1995).

An interesting possibility also exists for the inactivation

480 S. A. Khan


of the Rep protein of the archaeon plasmid pGT5, which

belongs to the pC194 family. The pGT5 initiator, Rep75,

has a nucleotidyl terminal transferase (NTT) activity that is

capable of transferring an A (or dA) residue to the 3 0 OH

end of a plasmid molecule that has been nicked by the

Rep protein (Marsin and Forterre, 1999). It has been pro-

posed that subsequent nicking of this DNA by Rep75

might result in the attachment of the A residue to the

active tyrosine residue of Rep75, thereby inactivating this

protein. This possibility remains to be tested experimen-

tally but, if true, would reveal another mechanism by

which RC initiators can be prevented from being reused in

replication.

Lagging strand replication and its contribution to the

promiscuity of RC plasmids

Replication of the ssDNA released upon completion of

leading strand synthesis initiates from the plasmid sso

and is done solely by the host proteins (reviewed by

Gruss and Ehrlich, 1989; Novick, 1989; Khan, 1997; del

Solar et al., 1998). Based on sequence as well as struc-

tural similarities, at least four different types of ssos, ssoA,

ssoT, ssoW and ssoU, have been identified (Gruss and

Ehrlich, 1989; Novick, 1989). An interesting aspect of

lagging strand replication is that some ssos (ssoA and

ssoW) function effectively only in their native hosts,

whereas others (ssoU and ssoT) can support ss ! dsDNA

synthesis in a broad range of bacterial hosts. All the ssos

have extensive intrastrand basepairing that results in

folded structures, although the sequences of individual

members of various sso types are generally variable

(Gruss and Ehrlich, 1989; Novick, 1989; Kramer et al.,

1999). As expected, the ssos are strand and orientation

specific and must be present in a ss form to be active. All

ssos analysed so far contain ssDNA promoters that are

recognized by the host RNA polymerase that synthesizes

a short RNA primer for DNA synthesis (Kramer et al.,

1997; 1998; 1999). Some ssos, such as ssoW, may

support RNA polymerase-independent primer synthesis

to a limited extent (reviewed by Khan, 1997). Plasmid RC

replication is generally asymmetric, in the sense that

lagging strand synthesis does not usually begin until after

the leading strand has been fully synthesized. This results

from the fact that the ssos are usually located slightly

upstream of the dso and, hence, are not exposed in a ss

form until after the parental leading strand has been

almost fully displaced. An interesting aspect of RC

plasmids is that, although plasmids belonging to a

particular family have similarities in their initiators and

dsos, their ssos are not necessarily similar. In fact, similar

ssos can be found among individual members of different

plasmid families (Gruss and Ehrlich, 1989; Novick, 1989;

Khan, 1997). This suggests that the evolutionary origin of

ssos is different from that of initiator protein/dso pairs, and

ssos may have been acquired by RC plasmids indepen-

dently of their leading strand replication elements. Some

plasmids contain up to three different ssos, although a

single sso is likely to function during ss ! ds replication in

an individual molecule. Interestingly, plasmids such as

pMV158 and pUB110 contain both ssoA- and ssoU-type

origins (del Solar et al., 1998). As ssoU can support

replication in different hosts, the benefit of also having an

ssoA in these plasmids is not clear at present. In plasmids

containing multiple ssos, deletion of one sso still allows

lagging strand synthesis from the other ssos in a

particular host. An issue that has so far not been resolved

is whether a particular sso is functionally dominant in a

plasmid containing multiple ssos. Although it is likely that

the sso that interacts most efficiently with the host RNA

polymerase will predominate (see below), this remains to

be tested.

Recent studies have provided significant insights into

the mechanism of lagging strand synthesis initiating from

the ssoA- and ssoU-type origins. The ssoA-type origins

found in different plasmids have a similar folded structure,

but include both conserved and variable sequences

(Gruss and Ehrlich, 1989; Novick, 1989). Two of the con-

served sequences, RSB and CS-6, have been shown to

be critical for RNA polymerase binding and termination of

primer RNA synthesis respectively (Kramer et al., 1997).

The RNA polymerase synthesizes short RNA primers

(approximately 17±18 nucleotides long) from the ssoA.

These primers are used by DNA Pol I for limited extension

synthesis, followed by replication of the lagging strand by

the more processive DNA Pol III. It is well known that

ssoAs are fully functional only in their native hosts. What

is the molecular basis for this host specificity? Preliminary

studies suggest that the strength of the interaction

between the ssoA and the host RNA polymerase may

determine, at least in part, its functionality in a particular

host (Kramer et al., 1998). In support of this hypothesis, it

has been shown that the S. aureus RNA polymerase

binds with much higher affinity to the ssoA of pE194 that is

native to S. aureus than to the ssoA of the streptococcal

plasmid pLS1 (Kramer et al., 1998). Interestingly, the ssoAs

can be specifically recognized for primer RNA synthesis

by a heterologous RNA polymerase, but this process is

presumably not very efficient (Kramer et al., 1998). It is

possible that specific nucleotides within the ssoA are

critical for their recognition by their cognate RNA poly-

merases, and resolution of this issue will require further

mutational and biochemical analysis.

In contrast to the ssoAs, the ssoU- and ssoT-type

origins can function in a variety of Gram-positive hosts.

What is the molecular basis for their broad-host-range

function? The ssoU that can function efficiently in both S.

aureus and B. subtilis has been shown recently to bind to



RNA polymerases from both of these organisms and

directs the synthesis of an approximately 45 nucleotide

long primer RNA (Kramer et al., 1999). Thus, the ability of

ssoU to be efficiently recognized by different RNA poly-

merases might explain, at least in part, its broad-host-

range function. It is possible that other host factors are

also important in broad-host-range function of the ssoU,

and the availability of purified replication proteins for

lagging strand synthesis in the future should allow further

investigation of this issue. It is known that the leading

strand replication of many RC plasmids is generally not

affected in heterologous Gram-positive bacteria (Khan,

1997). Therefore, the ability of a particular sso to function

in different hosts might be critical for the maintenance,

and therefore the horizontal spread, of drug-resistant RC

plasmids in bacteria.

As described earlier, all ssos have extensive secondary

structures (Gruss and Ehrlich, 1989; Novick, 1989; Khan,

1997; del Solar et al., 1998). An alignment of the various

sso sequences shows that the ssoT of plasmid pBAA1,

which is fully active in both S. aureus and B. subtilis, is

69% identical to the ssoU (Fig. 2). On the other hand, the

ssoAs of plasmids pLS1 and pE194, and the ssoW of

pWV01, which function only in their native hosts, have

50%, 52% and 59% identity, respectively, with the ssoU

(Fig. 2). Sequences conserved between ssoU and ssoT

but absent in the ssoAs and ssoW might be critical for the

broad-host-range replication and promiscuity of RC plas-

mids. It is interesting to note that sequences homologous

to RSB present in the ssoA-type origins are also con-

served in ssoU, ssoT and ssoW (Fig. 2). Thus, it is possible

that, although RSB is critical for RNA polymerase recog-

nition and initiation of primer RNA synthesis from all the

ssos, additional sequences present in ssoU and ssoT

(and absent in ssoAs and ssoW) stabilize their interaction

with the RNA polymerases from different hosts. Further

mutational and biochemical analyses are required to

resolve this interesting issue, which has implications for

the spread of drug-resistant RC plasmids in nature.

Regulation of RC replication

The regulation of plasmid replication, including that of RC

plasmids, is reviewed briefly in the accompanying article

by del Solar and Espinosa, this issue pp. 492±500, and

will not be discussed here in detail. In general, the Rep

proteins are rate limiting for the replication of RC plasmids

(Novick, 1989; 1998; del Solar et al., 1998). For plasmids

of the pT181 family, initiator synthesis is regulated at the

level of transcriptional attenuation of the Rep message by

an antisense RNA (del Solar et al., 1998). In the case of

the pLS1 plasmid, an antisense RNA as well as a

transcriptional repressor protein are involved in the

regulation of the Rep protein levels (del Solar et al., 1998).

Summary and perspectives

When plasmids that replicate by an RC mechanism were

originally identified in the mid-1980s, they were a mere

curiosity compared with their much better studied cousins,

the ssDNA bacteriophages of E. coli. Although the basic

process of RC replication in phages and plasmids has

been conserved, it is evident that RC plasmids have

evolved unique mechanisms that allow their replication to

be tightly regulated. One reason for this is the significant

difference in the biological activities of their initiator pro-

teins. Studies over the past several years have resulted in

an impressive body of information, including information

Fig. 2. Comparison of the ssoA, ssoU, ssoT and ssoW sequences found in various RC plasmids. The sequences were aligned using theCLUSTALW multiple sequence alignment software (Kramer et al., 1999). The sequences conserved in either four or all five ssos shown areshaded. The underlined nucleotides correspond to those in ssoU that are bound by the RNA polymerase (Kramer et al., 1999). The conservedRSB sequence and the proposed 210 and 235 sequences of ssoA (Kramer et al., 1997) are indicated. Boxed regions represent theconserved CS-6 sequences found in ssoA or their homologues in the other sso types.

482 S. A. Khan


on the basic replicon structure of RC plasmids, character-

ization of the biochemical activities of their Rep proteins,

interaction of the Rep proteins with the dso and identifi-

cation of the domains of dsos that are critical in the

initiation and termination of leading strand replication.

Furthermore, several groups of ssos with either a narrow

or a broad host range have been characterized, and it has

been suggested that the ssos may play an important role

in the spread of antibiotic-resistant plasmids and plasmid

promiscuity. However, studies on the nature of the

replication initiation complex that assembles at the dso,

the events that promote the termination of plasmid RC

replication and the molecular basis for the narrow- or

broad-host-range function of the plasmid ssos are in their

infancy. A critical gap in our knowledge of plasmid RC

replication also includes a lack of any structural informa-

tion on the initiator proteins and an understanding of how

their interaction with the dso results in structural changes

in the DNA that promotes the formation of an initiation

complex. Furthermore, a purified in vitro system for plas-

mid RC replication, which has not yet been developed, is

critical for an in-depth understanding of the molecular

events involved in the initiation and termination steps.

Such studies are likely to be the focus of intensive

research in the future.

Acknowledgements

I would like to thank members of my laboratory for helpfuldiscussions. The work in my laboratory has been supportedby Grant GM31685 from the National Institutes of Health.

References

Alonso, J.C., Stiege, C.A., Tailor, R.H., and Viret, J.-F. (1988)Functional analysis of dna (Ts) mutants of Bacillus subtilis:plasmid pUB110 replication as a model system. Mol GenGenet 214: 482±489.

Brown, D.R., Roth, M.J., Reinberg, D., and Hurwitz, J. (1984)Analysis of bacteriophage fX174 gene A protein-mediatedtermination and reinitiation of fX174 DNA synthesis. I.Characterization of the termination and reinitiation reac-tions. J Biol Chem 259: 10545±10555.

Bruand, C., and Ehrlich, S.D. (2000) UvrD-dependent repli-cation of rolling-circle plasmids in Escherichia coli. MolMicrobiol 35: 204±210.

Chang, T.-L., Kramer, M.G., Ansari, R.A., and Khan, S.A.(2000) Role of individual monomers of a dimeric initiatorprotein in the initiation and termination of plasmid rollingcircle replication. J Biol Chem 275: 13529±13534.

del Solar, G., Giraldo, R., Ruiz-Echevarria, M.J., Espinosa,M., and Diaz-Orejas, R. (1998) Replication and control ofcircular bacterial plasmids. Microbiol Mol Biol Rev 62: 434±464.

Dempsey, L.A., Birch, P., and Khan, S.A. (1992a) Uncouplingof the DNA topoisomerase and replication activities of aninitiator protein. Proc Natl Acad Sci USA 89: 3083±3087.

Dempsey, L.A., Birch, P., and Khan, S.A. (1992b) Six aminoacids determine the sequence-specific DNA binding andreplication specificity of the initiator proteins of the pT181family. J Biol Chem 267: 24538±24543.

Goetz, G.S., Englard, S., Schmidt-Glenewinkel, T., Aoyama,A., Hayashi, M., and Hurwitz, J. (1988) Effect of fX Cprotein on leading strand DNA synthesis in the fX174replication pathway. J Biol Chem 263: 16452±16460.

Gruss, A., and Ehrlich, S.D. (1989) The family of highlyinterrelated single-stranded deoxyribonucleic acid plas-mids. Microbiol Rev 53: 231±241.

Iordanescu, S. (1993) Identification and characterization ofStaphylococcus aureus chromosomal gene pcrA, identifiedby mutations affecting plasmid pT181 replication. Mol GenGenet 241: 185±192.

Jin, R., Zhou, X., and Novick, R.P. (1996) The inactive pT181initiator heterodimer, RepC/C*, binds but fails to inducemelting of the plasmid replication origin. J Biol Chem 271:31086±31091.

Khan, S.A. (1997) Rolling-circle replication of bacterial plas-mids. Microbiol Mol Biol Rev 61: 442±455.

Koepsel, R.R., and Khan, S.A. (1986) Static and initiatorprotein-enhanced bending of DNA at a replication origin.Science 233: 316±1318.

Koepsel, R.R., Murray, R.W., Rosenblum, W.D., and Khan,S.A. (1985) The replication initiator protein of plasmid pT181has sequence-specific endonuclease and topoisomerase-like activities. Proc Natl Acad Sci USA 82: 6845±6849.

Koepsel, R.R., Murray, R.W., and Khan, S.A. (1986)Sequence-specific interaction between the replication initia-tor protein of plasmid pT181 and its origin of replication.Proc Natl Acad Sci USA 83: 5484±5488.

Kramer, M.G., Khan, S.A., and Espinosa, M. (1997) Plasmidrolling circle replication: Identification of the RNA polymerase-directedprimerRNA and requirement of DNA polymerase I forlagging strand synthesis. EMBO J 16: 5784±5795.

Kramer, M.G., Espinosa, M., Misra, T.K., and Khan, S.A.(1998) Lagging strand replication of rolling-circle plasmids:specific recognition of the ssoA-type origins in differentGram-positive bacteria. Proc Natl Acad Sci USA 95:10505±10510.

Kramer, M.G., Espinosa, M., Misra, T.K., and Khan, S.A.(1999) Characterization of a single- strand origin, ssoU,required for broad host range replication of rolling-circleplasmids. Mol Microbiol 33: 466±475.

Marsin, S., and Forterre, P. (1999) The active site of therolling circle replication protein Rep75 is involved in site-specific nuclease, ligase and nucleotidyl transferaseactivities. Mol Microbiol 33: 537±545.

Muller, A.K., Rojo, F., and Alonso, J.C. (1995) The level ofthe pUB110 replication initiator protein is autoregulated,which provides an additional control for plasmid copynumber. Nucleic Acids Res 23: 1894±1900.

Noirot-Gros, M.F., Bidnenko, V., and Ehrlich, S.D. (1994)Active site of the replication protein of the rolling circleplasmid pC194. EMBO J 13: 4412±4420.

Noirot-Gros, M.-F., and Ehrlich, S.D. (1996) Change of acatalytic reaction carried out by a DNA replication protein.Science 274: 777±780.

Novick, R.P. (1989) Staphylococcal plasmids and theirreplication. Annu Rev Microbiol 43: 537±565.



Novick, R.P. (1998) Contrasting lifestyles of rolling-circlephages and plasmids. Trends Biochem Sci 23: 434±438.

Petit, M.-A., Dervyn, E., Rose, M., Entian, K.-D., McGovern,S., Ehrlich, S.D., et al. (1998) PcrA is an essential DNAhelicase of Bacillus subtilis fulfilling functions both in repairand rolling-circle replication. Mol Microbiol 29: 261±273.

Rasooly, A., and Novick, R.P. (1993) Replication-specificinactivation of the pT181 plasmid initiator protein. Science262: 1048±1050.

Soultanas, P., Dillingham, M.S., Papadopoulos, F., Phillips,S.E.V., Thomas, C.D., and Wigley, D.B. (1999) Plasmidreplication initiator protein RepD increases the processivityof PcrA DNA helicase. Nucleic Acids Res 27: 1421±1428.

te Riele, H., Michel, B., and Ehrlich, S.D. (1986) Are

single-stranded circles intermediate in plasmid DNA repli-cation? EMBO J 5: 631±637.

Thomas, C.D., Nikiforov, T.T., Connolly, B.A., and Shaw,W.V. (1995) Determination of sequence specificity betweena plasmid replication initiator protein and the origin ofreplication. J Mol Biol 254: 381±391.

Van Mansfeld, A.D.M., van Teefelen, H.A.A.M., Baas, P.D.,and Jansz, H.S. (1986) Two juxtaposed tyrosyl-OH groupsparticipate in X174 gene A protein catalyzed cleavage andligation of DNA. Nucleic Acids Res 14: 4229±4238.

Zhao, A.C., Ansari, R.A., Schmidt, M.C., and Khan, S.A.(1998) An oligonucleotide inhibits oligomerization of arolling-circle initiator protein at the pT181 origin of replica-tion. J Biol Chem 273: 16082±16089.

484 S. A. Khan


plasmid rolling-circle replication: recent developments

Documents