plasmid rolling-circle replication: recent developments
TRANSCRIPT
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
Q 2000 Blackwell Science Ltd, Molecular Microbiology, 37, 477±484
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
Q 2000 Blackwell Science Ltd, Molecular Microbiology, 37, 477±484
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
Plasmid rolling-circle replication 481
Q 2000 Blackwell Science Ltd, Molecular Microbiology, 37, 477±484
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
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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.
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