(Received for publication, August 8, 1994; and in revised form, November 2, 1994)
From the
Asymmetric rolling circle replication of the promiscuous replicon pMV158 is initiated by the plasmid-encoded RepB protein. In vitro, purified RepB protein introduces a nick within the leading strand origin of replication by a nucleophylic attack on the phosphodiester bond at the dinucleotide GpA. Some changes within and around this dinucleotide were recognized by the protein. RepB nicked and closed supercoiled pMV158 DNA, having an optimum activity at 60 °C. We have imitated, in vitro, a process of rolling circle replication, since RepB was able to nick (initiation) and to covalently close (termination) single-stranded oligonucleotides containing the protein cleavage sequence. Covalent DNA-protein complexes were not found, indicating that RepB has unique features among plasmid-encoded proteins involved in rolling-circle replication or conjugative mobilization.
Single-stranded coliphages and many small multicopy plasmids
replicate through an asymmetric RC ()mechanism, which is
characterized by the uncoupling of the leading and lagging strand
synthesis (Baas and Jansz, 1988; Novick, 1989; Gruss and Ehrlich, 1989;
del Solar et al., 1993a). The current model for plasmid
leading strand replication is mainly based on pT181 and related
plasmids (Rasooly and Novick, 1993; Rasooly et al., 1994) and
involves various stages: (i) a dimer of the plasmid-encoded initiator
of replication (Rep) protein binds to the plasmid DNA at a specific
region in the double strand origin, dso (Wang et al., 1993); (ii) one Rep subunit cleaves the
plasmid (+) strand by introducing a thyrosil phosphodiester
covalent bond at a specific DNA sequence (Thomas et al.,
1990); (iii) the Rep-generated nick leaves a free 3`-OH end, which is
extended by host proteins that use the(-) strand as a template
and displace the parental nicked (+) strand; (iv) the replisome
reaches the reconstituted dso and extends several more nt; and
(v) the newly synthesized strand is closed by the unused Rep monomer
through at least three nucleophylic DNA-protein-DNA attacks, leaving a
short oligonucleotide covalently bound to the attacking Rep monomer.
Thus, the final DNA products of plasmid leading-strand replication are
one dsDNA molecule and one ssDNA intermediate (te Riele et
al., 1986). One distinctive feature of this model is that the Rep
protein cannot be reused after each round of replication since the
final protein configuration is a heterodimer composed of an intact Rep
moiety and an inactive Rep-oligonucleotide subunit (Rasooly and Novick,
1993).
Plasmid pMV158 is a 5536-bp multicopy natural promiscuous
replicon that is the prototype of a family of RC-plasmids isolated from
several eubacteria (del Solar and Espinosa, 1992; del Solar et
al., 1993a). Various genes have been identified as being involved
in conjugative mobilization (mob), tetracycline resistance (tet), and replication and its control (repB, copG, rnaII; Fig. 1). This latter region has
been characterized in some depth for plasmid pLS1 (a mob derivative of pMV158; del Solar et al., 1993a, and
references therein). The Rep proteins of the pMV158 family share
conserved motifs with plasmid-encoded Tra and Mob proteins as well as
with Rep proteins of RC-plasmids, ss coliphages, and geminiviruses
(Pansegrau and Lanka, 1991; Ilyina and Koonin, 1992). However, the DNA
sequence at the dso of the pMV158-plasmid family has no known
homology with other replicons (del Solar et al., 1993a).
Initiation of pMV158 replication is mediated by RepB protein (24.2
kDa), which has been shown to introduce a specific nick between nt 448
(G) and 449 (A) of the complete nucleotide sequence of pMV158 (de la
Campa et al., 1990, Priebe and Lacks, 1989). Supercoiled DNA
is the only productive substrate for replication of pMV158 in a
cell-free extract (del Solar et al., 1987), and the nick site
is located on the loop of one of the major plasmid secondary
structures, Hairpin I (Puyet et al., 1988). This Hairpin I is
located within a region (termed nic) physically distant of the
RepB-binding site (del Solar et al., 1993a). In
vitro, RepB protein binds to a set of three directly repeated
sequences or iterons (the bind region) located 84 bp
downstream of the nick site (de la Campa et al., 1990; del
Solar et al., 1993a).
Figure 1:
Features
of pMV158. A, transcription map showing promoters (triangles), mRNAs, and antisense RNAs (wavylines) with the direction of transcription indicated (arrowheads). The region missing in pLS1 is indicated. The
origins of leading (dso) and lagging (sso) strands
are shaded. Synthesis of RepB is regulated at the
transcriptional level by the copG gene product. Both copG and repB are co-transcribed from promoter P. CopG binds to a plasmid region, which includes
the -35 region of P
, thus repressing its
own synthesis and synthesis of RepB (del Solar et al., 1990).
At the translational level, the amount of productive repB mRNA
could be regulated by the binding of the antisense RNA II to a region
in the cop-rep mRNA, which includes the repB initiation of translation signals (del Solar and Espinosa, 1992). B, the minimal dso of pMV158 as defined by deletion
analysis. Right and leftborders of the
regions missing in plasmids pLS1
24 (rightborder) and pLS1
A4 (leftborder)
are indicated. Positions of Hairpin II, Hairpin I, and the RepB-nick
site (
) are also shown. The RepB-generated footprints (
),
determined on linear dsDNA, are indicated on both strands. Downstream
of the nick site, the three 11-bp iterons (boldface, underlined) are located. These iterons are the in vitro determined RepB-binding site (de la Campa et al., 1990).
Other indicated features are the promoter for copG and repB genes (P
), the initiation site of
the cop-rep mRNA, the copG Shine-Dalgarno sequence (underlined), and the CopG initiation codon (boldface). C, nucleotide frequency distribution at
the dso of pMV158 between coordinates 400 and 500. Note that
the RepB-nick site (arrow) lies within a G+C-rich central
region, which is flanked by sequences with high A and T content,
suggesting that this region has a high potential to generate secondary
structures.
In the present work we show that, in vitro, purified RepB protein relaxed supercoiled pMV158 DNA
in a topoisomerase I-like reaction, having an optimal activity at 60
°C. The protein was also able to nick and to covalently join two ss
oligonucleotides containing the RepB-nicking sequence. These reactions
reproduce initiation (nicking) and termination (closing) of RC
replication. In addition, we have defined the bases involved in the
cleavage mediated by RepB. On linear DNA, RepB absolutely required the
nicking region to be in a single-stranded configuration. We have been
unable to isolate any RepB-oligonucleotide covalent intermediates, and
filter-binding assays showed that the RepBDNA complexes were
sensitive to treatment with SDS. We propose that a RepB
DNA
association, different than a stable covalent linkage, is involved in
the plasmid initiation of replication, similar to that of the gpII
protein of coliphage fd (Meyer and Geider, 1979). This is the first
example of a plasmid-encoded protein able to catalyze a site-specific
nicking reaction without forming a stable protein-DNA covalent linkage.
We had observed that the incubation of supercoiled pLS1
DNA with purified RepB protein at 37 °C resulted only in partial
relaxation of the plasmid, even at high protein/DNA ratios or prolonged
incubations (de la Campa et al., 1990). Plasmid pLS1
replicates in the mesophyle Bacillus subtilis, and a moderate
increase in its copy number has been observed when the cells are grown
at 45 °C instead of at 37 °C. ()We incubated
supercoiled pMV158 DNA with purified RepB protein at 37, 45, and 60
°C (Fig. 2A). Treatment of supercoiled (form FI)
DNA with RepB specifically yielded a mixture of nicked (FII) and
covalently closed relaxed (FI`) plasmid molecules, which reflect,
respectively, the nicking and nicking-closing ability of RepB.
Generation of forms FII+FI` was RepB-dependent, since only a
slight amount of such forms was observed in samples not treated with
the protein (Fig. 2A). To rule out the possibility that
some contaminant adenylated DNA ligase was present in the RepB
preparation (which could account for the closing reaction; Lehman,
1974; Kornberg and Baker, 1992), plasmid DNA was incubated with various
amounts of nicotinamide mononucleotide in the presence or absence of
RepB. The results (Fig. 2A) showed no significant
influence of this compound on the nicking-closing reaction, which
allows us to conclude that RepB is able to relax and to reseal
supercoiled pMV158 DNA. At a fixed time, and at a constant
concentration of RepB and its substrate, the amount of FII+FI` DNA
increased with the temperature, varying between 60% (at 37 °C) and
95% (at 60 °C). At 32 °C or below, less than 25% of the input
DNA was affected by incubation with RepB (not shown). A time-course
experiment showed that the RepB-mediated nicking (and nicking-closing)
was very fast at 60 °C, since about 80% of the substrate DNA was
converted to FII+FI` molecules after 1 min of incubation (Fig. 2B). The reaction slowed down at 45 °C, and
no plateau was reached at 37 °C. Concentration dependence assays (Fig. 2C) indicated that a majority of the pMV158 DNA
was relaxed by RepB in a period of 30 min in the presence of 34 ng of
the protein (or below, depending upon the temperature). Similar results
were obtained when supercoiled pLS1 plasmid DNA was used as substrate
(not shown). In the above experiments, the ratio of RepB molecules to
DNA molecules was 9.5:1, assuming that all protein molecules in our
RepB preparation were active, and that the active species of the
protein is a monomer. However, analyses of the RepB-configuration by
analytical ultracentrifugation showed that RepB is an hexamer (not
shown). The activity of RepB depended on the presence of
Mn
, this cation being partially replaceable by
Mg
(de la Campa et al., 1990). Incubation
with the topoisomerase I-inhibitor camptothecin (Avemann et
al., 1988), or with the pMV158-encoded CopG protein (del Solar et al., 1990), did not affect the RepB-mediated reactions (not
shown). Relaxation of supercoiled DNA by RepB did not seem to require
high energy cofactors, since incubation with ATP did not influence the
reactions. No consensus ATP-binding domain has been found in RepB.
Figure 2:
Nicking of pMV158 supercoiled DNA by
purified RepB protein. A, supercoiled pMV158 DNA (700 ng) was
incubated or not with purified RepB protein (45 ng; protein/DNA ratio
of 9.5:1 in molecules), in the presence or absence of the indicated
amounts of nicotinamide mononucleotide (BNMN) at the indicated
temperatures for 30 min. Samples were run in agarose gels with ethidium
bromide. Gels were photographed, and the negatives were scanned to
quantify the results. The main observed plasmid forms are supercoiled (FI), open circular (FII), and closed relaxed circles (FI`). Note that the RepB-untreated samples contained some FII
forms. (+) and(-) indicate the presence or absence of
protein, respectively. M, molecular weight standards. B, time-course of the nicking-closing reaction on supercoiled
pMV158 DNA by purified RepB protein. C,
concentration-dependence of the nicking-closing reaction on supercoiled
pMV158 DNA (700 ng/sample) by purified RepB protein. Reactions were
performed at 37 (), 45 (
), and 60 °C
(
).
Figure 4:
Sequence specificity of RepB. The activity
of purified RepB protein was assayed either on a 23-mer single-stranded
oligonucleotide containing the wild-type (wt) pMV158-nic region (5`-GCTACTACG/AC
-3`) or on 23-mer
having the indicated base changes (boxed). The
oligonucleotides (1.3 pmol) were labeled at its 5`-end with T4
polynucleotide kinase and were incubated with RepB protein (9.3 pmol)
for 30 min at 37, 45, and 60 °C. Protein/DNA ratio (in molecules)
was 7.3:1. Reaction products were separated on sequencing gels and
directly quantified. The amount of nicked DNA in each case was compared
with that obtained for the wt oligonucleotide. Oligonucleotide comp contains the nucleotide sequence complementary to the wt. A ds oligonucleotide made by hybridization of the wt and the comp 23-mers was not nicked by RepB (not
shown).
Figure 3:
Nicking activity of RepB on ssDNA. A, the 23-mer single-stranded oligonucleotide containing the
wild type nic region of plasmid pMV158 (oligowt: 5`-GGGGGGGCTACTACGACCCCCCC-3`) was labeled at its
5`-end with T4 polynucleotide kinase. 2.5 pmol of the labeled oligo was
treated with 9.3 pmol of RepB (ratio protein/DNA of 3.7:1, molecules)
at the indicated temperatures. The upper band (arrow) is the
23-mer oligonucleotide, the band below it (arrow) and the
other bands are, most likely, products from defective oligonucleotide
synthesis or of intrastrand pairing. They are also cleaved by RepB. The lowerband is the 15-mer cleavage product, which
co-migrates with a labeled band. B, the 23-mer single-stranded
oligonucleotide 5`-TGCTTCCGTACTACGACCCCCCA-3` (oligoC, Fig. 4), which lacked any possible intrastrand
pairing, was 5`-end labeled and treated with RepB, as in panelA, except that the amount of RepB was increased to show
maximum nicking. Note that no bands migrating between the 23- and the
15-mer products are visible in this case. M, ladder marker. C, The labeled oligo wt 23-mer (3.7 pmol) was incubated during
30 min, at 37 (), 45 (
), and 60 °C (
) with
various concentrations of RepB (3.7, 7.5, 11.2, 15, 18.5, and 28 pmol).
The protein/DNA ratios (in molecules) were of 1:1, 2:1, 3:1, 4:1, 5:1,
and 7.5:1, respectively. As controls, RepB-untreated samples were run.
The band co-migrating with the 15-mer RepB-product was considered as
control and subtracted from the RepB-treated samples after direct
quantification of the RepB products.
The sequence specificity of RepB on the
nick site was tested by assaying its nicking activity on
single-stranded 23-mer oligonucleotides in which base changes at the
RepB-nick region were introduced. The reaction products were quantified
and compared with the RepB nicking activity on the 23-mer containing
the wild-type nick site. As can be observed (Fig. 4), at 5` of
the nick site (G/A
) only a
G
A change was allowed, although it reduced by 50%
the amount of digested DNA. At 3` of the nick, the A
could
be changed by C or T without much reduction in the RepB activity.
However, one G at this position made the oligonucleotide insensitive to
RepB. In addition, a C to G change in the second nt after the nick site
(C
G) resulted in a substrate not recognized by RepB.
This finding was unexpected because we had ascribed the C
tract at 3` of the nick site as being mainly involved in the
generation of Hairpin I on supercoiled DNA, by intrastrand pairing with
the G
tract 5` of the nick. Generation of this structure on
supercoiled DNA would indeed expose the phosphodiester bond in the GpA
nick site as a single-stranded region. The failure of RepB to nick the
23-mer containing the C
G change, could indicate
either a requirement for the generation of a secondary structure in the
ssDNA substrate or that the nt at this particular position is
important. The latter explanation is more likely, since (i) the oligo B
(having the C
G change) could generate a secondary
structure but was still insensitive to RepB and (ii) the oligo C is
fully sensitive to RepB and yet it shuld not generate any intrastrand
pairing. In addition, changing the C-stretch downstream of this
C
nt by A and T residues did not affect cleavage by RepB
(not shown). The protein did not recognize the sequence complementary
to its nicking region (oligo comp), which demonstrates that
nicking by RepB is sequence-specific. This oligonucleotide also
contains the G
and C
ends, so that intrastrand
pairings and secondary structures within the RepB substrate are not
important features when the substrate is in a single-stranded
configuration. We conclude that C
also plays an important
role within the DNA sequence for RepB nicking. The critical role of
sequences downstream of the nick site for RepB-cleavage contrasts with
the requirements observed for TraI protein of plasmid RP4, which
requires 6 nt upstream of the nick site but has no base specificity
downstream of it (Pansegrau et al., 1993). Finally, a ds
oligonucleotide containing the wild type nicking region, obtained by
hybridization of the oligonucleotides wt and comp, was not a substrate
of purified RepB protein (not shown). Since RepB relaxed supercoiled
DNA (Fig. 2) but did not cleave linear dsDNA, it would appear
that the nic region has to be exposed in a single-stranded
configuration within the supercoiled DNA plasmid molecules. In fact,
RepB was unable to nick linear ds pLS1 DNA as judged from alkaline
agarose gels. (
)RepB protein specifically cleaved ssDNA
harboring some changes in the nic region (Fig. 4),
suggesting that the protein could recognize as substrate other dso of pMV158-related plasmids. We have preliminary evidence
suggesting that this is, indeed, the case. We can then draw the
following conclusions: (i) the DNA sequence cleaved by RepB has to be
in a single-stranded conformation; (ii) generation of a secondary
structure within the 23-mer is not required; and (iii) the protein
recognizes the 9-nt consensus sequence (5`-TACTACR/HC
(IUB code, R = puryne; H = not G). A
computer search revealed that no other nt sequence of this kind is
present in pMV158.
Figure 5: Nicking-closing activity of RepB on ssDNA. The indicated 23- and 26-mer single-stranded oligonucleotides were labeled at their 5`-ends. Both oligonucleotides contain the RepB nick site (/) in a different position. Mixtures of labeled and unlabeled substrates were made and used as a substrate for RepB protein. For each reaction, the protein DNA ratio (in molecules) was of 0.3:1 or 0.6:1. The assays were performed at the indicated temperatures. The labeled nicking and nicking-closing products were visualized on sequencing gels; M, labeled oligonucleotide mixtures used as markers. The expected reaction products are indicated on the top of the figure.
The closing reaction mediated by RepB was further analyzed by attempts to join oligonucleotides that contained, respectively, the right and the left half of the nick region (Fig. 6). First, a 5`-labeled 15-mer ending at the nick site (pG-OH) was mixed with a phosphorylated 8-mer initiating at the nick site (pAp). If RepB were able to covalently join both halves of its nick region without previous nicking, a 23-mer reaction product should be detected. The results (Fig. 6A) showed that this was not the case, indicating that the substrate has to be cleaved by the protein prior to the closing reaction. To test this, the same labeled 15-mer (ending in pG-OH) was mixed with a labeled 26-mer oligonucleotide, which contains all of the wild-type nicking region, and treated with RepB (Fig. 6B). In this case, the protein cleaved the 26-mer to generate a labeled 12-mer product. In addition, a new band corresponding to a labeled 29-mer oligonucleotide was also identified. The new 29-mer product should arise from the RepB-mediated covalent join of the nicked 3`-half (14-mer) with the 3`-OH end of the 15-mer. This closing reaction was not observed if the 15-mer to be joined has an end different than the wild-type, even if the pG-OH was kept unchanged (not shown). A complementary experiment was performed; the above 26-mer was mixed with a labeled 8-mer containing the region 3` to the nick and was incubated with RepB. The 20-mer closing product was not detected (Fig. 6C). Taking these two results together, we conclude that the protein can only close the nick if a RepB-generated 5`-phosphate end is provided. The 3`-OH end to be closed has to belong to the nick sequence, but it does not need to be generated by the protein. These results indicate that the RepB-mediated nicking reaction would leave an ``activated'' 5`-phosphate end 3` to the nick. Since RepB failed at nicking oligonucleotides extracted from polyacrylamide gels, verification of this putative activation by reisolation of the 14-mer RepB-generated intermediate (Fig. 6B) was not feasible. In addition, we have observed that gel retardation assays performed with RepB and the 26-mer oligonucleotide did not generate any retarded band (not shown), perhaps indicating that protein-DNA complexes were dissociated during electrophoresis. Whether ``activation'' is the consequence of a reaction that requires the simultaneous presence of the protein and the two substrates to be joined (the 26- and the 15-mer, Fig. 6B) and whether this reaction could be somehow uncoupled is under investigation.
Figure 6: RepB-mediated closing reactions. Schematic representations of the nicking-closing experiments performed with single-stranded oligonucleotides, and the expected results after RepB-treatment are depicted at the top of each panel. The reaction products are shown at the bottom. The substrates employed were the oligonucleotides indicated in Fig. 5, labeled at their 5`- or 3`-ends (indicated by *) or unlabeled. The oligonucleotides were treated (+) or untreated(-) with RepB at the indicated incubation temperatures. The lengths of the detected oligonucleotides are indicated at the sides of the different autoradiograms. A, the 5`-labeled 15-mer, ending in pG-OH, was mixed with the unlabeled 8-mer starting in pA. Note that no 23-mer closing product is observed. B, the 5`-labeled 26-mer containing the GpA RepB-nicking site was mixed with the labeled 15-mer ending in pG-OH. Note the appearance of the 29-mer RepB-generated product. C, the above 26-mer was mixed with the phosphorylated 5`-labeled 8-mer (starting in pA). The 20-mer closing product was not detected. D, the 23-mer was 3`-end labeled, treated with RepB, and the reaction products were digested with Proteinase K. A mixture of phosphorylated and nonphosphorylated 5`-labeled 8-mer was run; the latter migrates slower than the former. Generation of a tyrosylphosphodiester covalent bond between RepB and its substrate should yield a peptide-DNA adduct having an electrophoretic mobility different from that of the free phosphorylated 8-mer.
Several nicking-closing
enzymes (Rep protein of the RC-plasmid pT181, gene A product from
X174, and Tra and Mob proteins from plasmids RP4 and RSF1010) are
able to nick and close DNA through a stable covalent protein-DNA
intermediate (Rasooly and Novick, 1993; Langeveld et al.,
1978; Pansegrau et al., 1990; Scherzinger et al.,
1992). To know if a covalent RepB-DNA intermediate is generated, the
23-mer oligo wt (Fig. 4) was labeled at its 3`-end and treated
with RepB protein. The reaction products were digested with Proteinase
K in order to leave a short peptide linked to the DNA if a covalent
protein-DNA complex were generated. As can be seen (Fig. 6D), only free DNA species of 8-mer in size (in
addition to the 23-mer) were detected. These are the expected products
if no covalent DNA-peptide heteromolecules are the reaction products.
Several other experiments aimed at detecting any possible RepB-DNA
covalently linked intermediates, using either supercoiled DNA or
single-stranded oligonucleotides as substrates, gave negative results
(not shown). To analyze the nature of the RepB
DNA complexes, we
made use of a filter-binding assay (de la Campa et al., 1990).
To this end, the 23-mer oligo wt was 3`-end labeled and incubated with
RepB at 60 °C, in conditions of maximum nicking (Fig. 3).
The reaction products were filtered, and the filters were washed with
various buffers (Table 1). The results showed that RepB
DNA
complexes were extremely sensitive to SDS, again supporting the
noncovalent nature of the RepB-binding to DNA. Complexes were
moderately sensitive to high salts, indicating that electrostatic
interactions take place between the protein and its target DNA. The
predicted isoelectric point of RepB is 8.8, so we should expect a
reduction in the protein
DNA complexes once this point is
surpassed. Washing buffers with pH above this point also reduced the
amount of DNA retained in the filters (Table 1), suggesting that
the interactions between RepB and DNA could take place through
positively charged amino acids. Based on all of the above data, we
tentatively conclude that RepB
DNA interaction can be exerted
either through the generation of a transient phosphodiester bond, as is
the case with the gene II product of coliphage fd (Meyer and Geider,
1979), or through a noncovalent binding.
The initiator of replication RepB protein from plasmid pMV158
has nicking-closing, sequence-specific activities, as expected for a
protein involved in RC-replication. We have simulated a process in
which purified RepB protein performed the reactions involved in
initiation and termination of plasmid RC-replication. RepB has specific
nicking activity on ssDNA containing the nic region, which is
the one present in the plasmid (+) strand. RC-replication shares
features with conjugative processes since ssDNA intermediates are
generated, and proteins involved in conjugative transfer also nick
their substrate DNA (Pansegrau et al., 1990, 1993). However,
in contrast to other Rep, Tra, and Mob plasmid-encoded proteins, which
generate a proteinDNA covalent bond (Thomas et al.,
1990; Pansegrau et al., 1990; Scherzinger et al.,
1992; Rasooly and Novick, 1993), RepB protein of pMV158 did not show
any detectable covalent link with DNA. RepB absolutely required
single-stranded or supercoiled DNA substrate to nick its target since
it failed to cleave a ds oligonucleotide containing the nicking site
and it was unable to nick linear ds pLS1 DNA.
In addition,
replication of pMV158 in a cell-free extract absolutely required
supercoiled DNA (del Solar et al., 1987). The RepB-requirement
for supercoiled substrate and its activity on ssDNA strongly indicate
that generation of Hairpin I and exposure of the nick site in a
single-stranded configuration is essential for initiation of
replication of pMV158 (del Solar et al., 1987, 1993b, this
work). The above features of RepB resemble those found for proteins A
and gpII from coliphages
X174 and fd respectively, since both
proteins also need the substrate to be supercoiled or single-stranded
(Langeveld et al., 1978; Meyer and Geider, 1979; van Mansfeld et al., 1980). Although negatively supercoiled DNA seems to be
the preferred substrate for nicking-closing proteins, the MobABC
complex of plasmid RSF1010 nicks linear dsDNA with identical
requirements as those for supercoiled DNA, except that more MobC is
needed (Scherzinger et al., 1992). Furthermore, RepC of
plasmid pT181 acts on supercoiled DNA (Noirot et al., 1990),
but it is able to conduct in vitro replication with a relaxed
DNA substrate (Khan et al., 1982) and shows specific nicking
activity on lineal dsDNA in vitro (Koepsel and Khan, 1987).
The reaction conditions of RepB also differ from other nicking-closing
enzymes. RepB is an hexamer; it shows an optimal temperature at 60
°C, and it requires Mn
, whereas the other
proteins in general have a dimeric configuration, they require
Mg
and show optimal temperature for cleavage at 37
°C or below.
Many of the Tra and Mob proteins exhibit common
motifs that are also conserved in the Rep proteins of RC plasmids and
ssDNA coliphages (Pansegrau and Lanka, 1991; Ilyina and Koonin, 1992;
Waters and Guiney, 1993). Furthermore, the dso of many
RC-plasmids, the (+) origins of ssDNA coliphages, and the oriT of conjugative plasmids have sequence homologies at their nic region (Waters and Guiney, 1993). Although the protein motifs are
conserved in the pMV158 plasmid family, the nic region in the
DNA of these plasmids has no resemblance with known cognate regions in
other plasmids or coliphages (del Solar et al., 1993a). Three
possible Tyr residues (at positions 14, 99, and 115) could be the
candidates for realizing the nucleophilic attack on the phosphodiester
bond at the RepB nick site by a tyrosil-OH group. The residue most
likely involved in this reaction is Tyr, since it is
conserved among all nicking-closing proteins from plasmids and viruses
(Ilyina and Koonin, 1992).
One feature of the plasmids of the pMV158
family is that they all contain two or three iterons of different
lengths, which are separated from the nick site by a spacer sequence
ranging between 13 and 91 bp (del Solar et al., 1993a). In the
case of pMV158, purified RepB protein binds to the iterons and
introduces the nick 84 bp upstream of its binding site (de la Campa et al., 1990). Nevertheless, with the substrates employed
here, RepB was able not only to nick and close pMV158 supercoiled DNA
but also linear ssDNA, which lacked the iterons. This behavior differs
from gpII protein of the filamentous phage f1, which requires the
presence of its binding region (in a ds configuration) to introduce the
nick within a single stranded oligonucleotide (Higashitani et
al., 1994). Although the protein did not generate a stable
covalent linkage to DNA, the 5`-end (but not the 3`-end) generated in
the nicking reaction was activated by RepB. The ability of RepB to join
the 5`-phosphate end of the nicking reaction product with a new 3`-end (Fig. 6B), in conjuction with the failure of the
protein to catalize the joining of the 3`-OH generated during nicking
with a provided 5`-phosphate end (Fig. 6C), indicated
that the 5`-end to be closed is activated somehow by the RepB nicking.
This RepB-mediated activation of the 5`-end suggests that the
proteinDNA interactions occurring at the initiation of
replication are important for the termination process, if this
activation is maintained during replication. We envisage termination of
pMV158 RC replication as the result of the following reactions: (i)
introduction of a second nick at the GpA dinucleotide, the A residue
belonging to the newly synthesized DNA and becoming activated (perhaps
by a new RepB molecule); (ii) a closing reaction between the pG-OH end
of the newly synthesized DNA and the A residue activated in this second
nicking reaction, with the concomitant release of a dsDNA molecule, and
(iii) a second closing reaction between the 5`-pA end, activated at the
initiation stage, and the pG-OH end generated in the second nicking
reaction, which leads to the release of a circular ssDNA molecule.
Activation could indicate that either the protein or the nicked DNA
suffered a conformational change to keep the energy to close the nick.
In our opinion, the first possibility seems more likely. However, as in
the case of gpII protein from filamentous coliphages, how the energy of
the RepB-cleavage is stored for the ligation reaction is unknown
(Kornberg and Baker, 1992). Consequently, the nicking-closing reaction
mediated by RepB more closely resembles that exerted by the gpII
protein of coliphage fd (Meyer and Geider, 1979) than those by Rep
proteins of the RC-plasmids of the pT181 family (Thomas et
al., 1990, Wang et al., 1992, Dempsey et al.,
1992; Rasooly et al., 1994). Whether RepB acts through a
noncovalent nucleophylic attack toward its target or through a
transient covalent bond, is not yet known.
To explain how RepB acts
at a distance, a complex DNA structure for this region was proposed (de
la Campa et al., 1990). A DNA intrinsic curvature appears to
exist within the plasmid origin, as reflected by (i) the presence of
(dT-dA) tracts spaced by integer helix turns (Fig. 1B), (ii) the anomalous migration of dso-containing DNA fragments in polyacrylamide gels (not
shown), and (iii) the distribution of bands of hipersensitivity to
DNase I, as well as the hydroxyl radical footprinting pattern of naked
DNA (de la Campa et al., 1990). Cylindrical projection of the
DNA helix between coordinates 400 and 600 (not shown) indicates that
the RepB nick site and the center of the iterons are located in the
same face of the helix. We propose that the DNA sequence between the
iterons and the nic region could provide a flexible arm
(flexibility, which could be increased by Mn
ions),
so that the binding of RepB to the iterons could appropriately place
the protein close to Hairpin I. Such a highly sophisticated
protein
DNA interplay should lead to local unwinding of the DNA
and to the exposure of the nick site in a single-stranded form. Whether
this spacer acts as an enhacer for replication, as is the case of
filamentous phages (Horiuchi, 1986), is not known. Be that as it may,
the actual mechanism of termination of replication by RepB is
intriguing. The elegant model proposed by Rasooly and Novick(1993) for
termination of pT181 replication is based on the inactivation of RepC
protein through generation of a RepC heterodimer. However, in the case
of RepB of pMV158, the lack of stable covalent binding of RepB to DNA
would indicate that this mechanism of inactivation does not occur in
pMV158. Yet, the plasmid copy number is subjected to a strict control
which, in addition to the role of the plasmid-encoded repressors (del
Solar and Espinosa, 1992; Fig. 1), should involve lack of
recycling of the initiator protein.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M28538[GenBank].