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1 Belarussian State University, Biological Faculty, Department of Genetics and Biotechnology, Minsk 220050, 4 Scorina Avenue, Belarus
2 Memorial Sloan-Kettering Cancer Center, NY 10021, USA
3 Laboratoire de Génétique Microbienne, Bâtiment des Biotechnologies, INRA, 78352 Jouy en Josas, France
Correspondence
Laurent Janniere
laurent.janniere{at}jouy.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1. Most importantly, this replication machinery contains the two type C polymerases, PolC and DnaE. As the mechanism of initiation of the three genomes is substantially different, this suggests that both type C polymerases might be required in any Cairns replication in B. subtilis and presumably in other bacteria encoding PolC and DnaE.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Giraldo, 2003; Leipe et al., 1999). Surprisingly, this diversity, obvious when comparing Eubacteria to Archaea and Eukarya, is also manifest among bacterial species. For instance, in the well characterized eubacteria Escherichia coli and Bacillus subtilis, the primosomes required for initiating replication at origins or collapsed replication forks have been shown to greatly differ in their constitution and mechanism of helicase loading (Bruand et al., 1995; Cox et al., 2000; Davey & O'Donnell, 2003; Lemon et al., 2002; Marsin et al., 2001; Polard et al., 2002; Velten et al., 2003). Differences have also been documented in the DNA polymerase III (Pol III) holoenzyme, a subassembly of the replication machinery where DNA synthesis is catalysed. In E. coli this element contains 10 different subunits of which four (
, 
, 
and 
) are missing in B. subtilis (Bruck & O'Donnell, 2000; Bruck et al., 2002; Lemon et al., 2002; Martinez-Jimenez et al., 2002; McHenry, 2003).
Based on systems for which a Pol III holoenzyme has been isolated and/or reconstructed in vitro, it is generally thought that both DNA strands of genomes are polymerized by a single replicative polymerase of the C (prokaryotes) or B (eukaryotes) families, dimerized in a holoenzyme structure (for reviews, see Benkovic et al., 2001; Bullock, 1997). However, this scheme might not be universal as replication in numerous organisms depends on two different polymerases. For instance, in B. subtilis and related bacteria of the Bacillus/Clostridium and Thermotogales groups, two type C polymerases (PolC and DnaE) are essential for chromosome replication and colocalize with the replication machinery (Dervyn et al., 2001; Inoue et al., 2001; Lemon & Grossman, 1998, 2000). In the replication fork of the B. subtilis plasmid pAM1, PolC might be mainly involved in leading strand synthesis while DnaE might be required for efficient lagging strand polymerization (Dervyn et al., 2001). This polymerase specialization detected in a plasmid system might also apply to chromosomal forks as (i) initiation of pAM1 replication depends on the cellular machinery that initiates DNA synthesis at collapsed replication forks (Bruand et al., 1995, 2001; Marsin et al., 2001; Polard et al., 2002) and (ii) a bioinformatic study showed that bacteria containing both PolC and DnaE have a much stronger gene strand bias than DnaE+ and PolC– species (Rocha, 2002). This latter point suggests that asymmetrical replication forks could have a major role in organizing bacterial chromosomes. DNA replication also depends on two different replicative polymerases (Pol
and Pol
, B family) in numerous eukaryotic cells (for reviews, see Burgers, 1998; Hubscher et al., 2000; Johnson & O'Donnell, 2005; Kawasaki & Sugino, 2001; Sugino, 1995). As in bacterial forks, these polymerases might be specialized as several data suggest that Pol would elongate leading strand synthesis while Pol would synthesize the lagging strand (see, for instance, Fukui et al., 2004; Garg et al., 2004; Karthikeyan et al., 2000; Morrison & Sugino, 1994; Shcherbakova & Pavlov, 1996). Recently, it was proposed that chromosomal replication in archaea might also depend on two distinct DNA polymerases (Henneke et al., 2005).
To further test the requirement for PolC and DnaE in Gram-positive bacteria, we undertook a genetic study of the replication of B. subtilis plasmid pBS72 (Titok et al., 2003). Results showed that pBS72 replication (i) is of the theta-type, (ii) depends on two plasmids elements, an initiator protein and an origin of replication, (iii) initiates by an original mechanism independent of the cellular initiators DnaA and PriA and (iv) is carried out by a replication fork indistinguishable from that of the B. subtilis chromosome, including PolC and DnaE. These data illustrate again how plasmids capture for their own benefit cellular factors and support our previous proposal that the catalytic centre of the B. subtilis replication machinery might include two different type C polymerases.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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); 1A74 (dnaX8132 hisA1 thr-5), 1A224 (hisH2 pheA1 trpC2) and 1A226 (polA5 hisH2 pheA1 trpC2) (Bacillus Genetic Stock Center); EDJ51 (168 dnaE2.10 ins-phl), EDJ58 (168 dnaE2.6 ins-phl), HVS609 (168 spac-I : : polC emr containing pMAP65) and HVS614 (168 spac-I : : dnaE emr containing pMAP65) (Dervyn et al., 2001); PB1669 (168 dnaF69) (Attolini et al., 1976); CRK6000 (purA16 metB5 hisA3 guaB) and NIS6200 (CRK6000 proA : : pBPA21 : : cat) (Hassan et al., 1997); NIS6311 [CRK6000 spoIIIJ : : pBPA21 : : cat dnaA(ochre)] (Shigeki Moriya, University of Technology, Sydney, Australia); HVS606 (168 pcrA3 ins of pMAP55 in pBR322-CmR inserted at the metD locus) (Petit et al., 1998); PPBJ120 (168 priA1 : : pAPJ11 : : erm) and PPBJ65 (168 priA : : pAPJ12 : : erm, PriA+) (Polard et al., 2002). Hosts for plasmid constructions were regular E. coli strains. Growth in a rich medium (LB), induction of competence and transformation were as described previously for B. subtilis (Bron, 1990) and for E. coli (Sambrook et al., 1989). To construct the 
priA strain, 168 competent cells harbouring pMTLBS72 (Titok et al., 2003) were transformed to erythromycin resistance with total DNA prepared from PPBJ120. Transformants were isolated and propagated in a poor medium (Polard et al., 2002). Antibiotics were used at the following concentrations (µg ml–1): ampicillin, 100; chloramphenicol, 6; erythromycin, 0·6; phleomycin, 2. To investigate the effect of PolC inhibition by the drug HB-EMAU (Wright et al., 2005) fresh cells harbouring pMTLBS72 were grown in the presence of various concentrations (0, 0·625, 1·25, 2·5 and 5 µM) of the drug for about 1·5 h. In these conditions, cell growth was slightly reduced at 1·25 µM and arrested at 
2·5 µM HB-EMAU (not shown). Filamentation was observed at 1·25 µM.
DNA manipulation and plasmid construction.
DNA preparation and manipulation were carried out essentially according to standard procedures (Bron, 1990; Sambrook et al., 1989). Previously described plasmids used were pMTL20C, pMTL21E (Swinfield et al., 1991) and pMTLBS72 (a pMTL20C derivative containing a BglII fragment carrying the pBS72 replicon; Fig. 1, top line) (Titok et al., 2003). Derivatives of pMTLBS72 carrying a truncated or mutated pBS72 replicon (Fig. 1) were generated in E. coli by incubating the parental plasmid with restriction enzymes cleaving in the cloned fragment and, when appropriate, in the flanking polylinker (segments were blunt-ended with the Klenow enzyme when required). The plasmids yielded were termed pLJH16-18 and pLJH67-76. The DnaA requirement was investigated with the pBS72 derivative pLJH13 composed of the pBS72 BglII fragment of pMTLBS72 cloned at the BglII site of the erythromycin-resistant E. coli vector pMTL21E.
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). CN values presented in Table 1 were calculated from three independent measurements. Control experiments showed that the integrated plasmid replicon (pLS32) that directs chromosomal replication in the dnaA strain (Hassan et al., 1997) does not affect pBS72 derivative CN values (not shown). CN measurement in the priA strain was carried out in LB with fresh transformants isolated in a poor medium. Since the strain is genetically unstable (Bruand et al., 2001; Polard et al., 2002), the PriA– phenotype was confirmed genetically at the same time as DNA preparation (not shown).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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90 kb) plasmids of a Belarus collection of B. subtilis strains (Titok et al., 2003). The segment, fused to a pUC-like plasmid to form pMTLBS72, contains three complete ORFs (Fig. 1
, top). The product of ORF-1 (hereafter termed RepA) might correspond to the plasmid initiator as it contains a putative DNA-binding motif (HTH motif between amino acids 111 and 132) and a C-terminal region (position 276–330) homologous (35 % identity, 63 % similarity) to the central part of the N-terminal domain I of the highly conserved chromosomal initiator DnaA. In E. coli, this internal region of domain I (position 13–68) is mainly involved in stable binding to the replicative helicase (Felczak et al., 2005; Seitz et al., 2000; Sutton et al., 1998). The 
400 bp downstream sequence would include the plasmid origin as it contains typical hallmarks of such replication elements: repeated sequences (iterons), DnaA-binding sites and an AT-rich region (for reviews, see del Solar et al., 1998; Espinosa et al., 2000; Helinski et al., 1996). The function of the remaining two ORFs, which have no homologues in databases, is not known.
To genetically identify plasmid sequences required for replication, pMTLBS72 derivatives carrying parts of the 3 kb replicon were constructed and assessed for replication activity in B. subtilis by a transformation assay. Results (Fig. 1) showed that the minimal segment capable of autonomous replication is 1929 bp in length (pLJH76). It encompasses repA and the downstream 400 bp region. As expected, the repA product is essential for replication as a 5' deletion of the coding frame and a truncation of the protein by frameshift mutations at the NsiI or SalI sites abolish transformation activity (pLJH16-18 and pLJH67-68). The 400 bp downstream sequence is also essential for replication since (i) its deletion (pLJH71 and pLJH75) causes a complete loss of plasmid replication and (ii) its alteration by a small (32 bp) deletion changing the spacing between two repeats (pLJH74) sharply reduces the transformation activity and the CN value of the replicon. These results suggest that RepA is the plasmid initiator and that the 400 bp includes the plasmid origin. Note that none of the functional derivatives (except pLJH74) displayed a marked change in CN (not shown). This indicates that ORF-2 and ORF-3 are not needed for replication control and that the CN of the replicon might be regulated at the level of repA expression and/or by origin handcuffing (del Solar et al., 1998; Espinosa et al., 2000; Helinski et al., 1996; Park et al., 2001).
Host-encoded replication functions
To search for host-encoded proteins required for plasmid replication, pMTLBS72 was introduced into wild-type and isogenic mutants affected in DNA polymerases PolC (dnaF69), DnaE (dnaETs2.6 and 2.10) or Pol I (polA5), the processivity factor DnaN (dnaG5), the replication machinery organizer DnaX (dnaX8132), the initiator proteins DnaA (dnaA) and PriA (priA), the replicative helicase DnaC (dnaC14), the DnaC loaders DnaB, DnaD and DnaI (dnaB19, dnaD23 and dnaI2), the primase DnaG (dnaE20) and the PcrA helicase required for rolling-circle replication and involved in Cairns replication (pcrA3) (for reviews, see Khan, 2005; Lemon et al., 2002; Moriya et al., 1999; Winston, 1982; Yoshikawa & Wake, 1993). Transformants were readily obtained at 30 °C with the complete set of strains. To measure the effect of T mutations on plasmid replication, total DNA was extracted before and after a short growth (about five generations) at the semi-restrictive temperature. The DNA was then restricted, run in agarose gels and analysed by Southern blotting and hybridization, using radiolabelled plasmid DNA and a chromosomal PCR fragment as probes. The ratios of plasmid/chromosomal signals were then determined before and after growth at the semi-permissive temperature and compared. In the case of null or constitutive mutations (dnaA, priA, polA5 and pcrA3), plasmid/chromosomal ratios were measured at 37 °C and compared to those of the isogenic wild-type strains grown at the same temperature. Replica-plating analysis showed that the plasmid was maintained in the cell population throughout the experiment in most of the mutants. However, for the dnaB19 and, to a lesser extent, dnaI2 mutants, cured cells were detected at the end of growth in proportions reaching 0·5 and 0·3, respectively. The proportion of plasmid-free cells was taken into account for CN determination.
In most mutants, a moderate (two- to threefold) CN reduction was observed at semi-restrictive temperature (Fig. 2 and Table 1). Plasmid replication is thus more affected than chromosome replication in these strains, showing that the corresponding mutated factors are required for pBS72 replication. This applies to the polymerases PolC and DnaE, the processivity factor DnaN, the replication machinery organizer DnaX and the primosomal proteins DnaB, DnaC, DnaD, DnaG and DnaI. In strains lacking DnaA or PriA (because of gene deletion) or encoding a replication-defective form of the polymerase Pol I (polA5 mutation; Laipis & Ganesan, 1972) or an inactive form of the PcrA helicase (pcrA3; Anand et al., 2005), the CN is either unchanged (Pol I and PcrA mutants) or increased (DnaA and PriA mutants). These factors are hence not required for pBS72 replication. As the DnaA and PriA null strains filament (Hassan et al., 1997; Polard et al., 2002; our observations), the CN increase observed in these contexts might result from dilution of negative factors in enlarged cells allowing plasmid amplification. However, in the DnaA context, it cannot be ruled out that the CN increase results from alleviation of a negative effect of DnaA on plasmid replication, because DnaA might bind to dnaA boxes of the plasmid origin or to host-encoded factors limiting for plasmid replication. Finally, it should be noted that (partial) inactivation of required replication factors did not always cause CN reduction. In several instances, the (partial) inactivation kept the CN unchanged. This occurs, for instance, when PolC inactivation is caused by the dnaF33 mutation or when cells are grown in the presence of the PolC-inhibitory compound HB-EMAU (not shown). A similar result was obtained in cells depleted of the wild-type PolC or DnaE enzymes because of the weak expression of the corresponding gene expressed from the IPTG-inducible spac-I promoter (not shown). This probably results from a simultaneous arrest of plasmid and chromosome replication. Finally, the lack of single-stranded production in any PolC and DnaE replication mutant precluded the determination of whether these enzymes are specialized in strand synthesis (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; this work (for reviews, see del Solar et al., 1998; Espinosa et al., 2000; Helinski et al., 1996)]. On the contrary, unlike rolling-circle-replicating plasmids, (i) pBS72 is independent of PcrA helicase activity, (ii) it lacks any homology with rolling-circle plasmids which are classified into a dozen groups based on sequence similarity, (iii) none of its derivatives accumulate single-stranded DNA as do generally derivatives of rolling-circle genomes and (iv) pBS72 is much larger (90 kb) than any rolling-circle plasmids (
10 kb) [Titok et al., 2003; this work (for reviews, see del Solar et al., 1998; Espinosa et al., 2000; Khan, 2005)].
The second aspect of replication uncovered by this study concerns the mechanism of initiation of pBS72 replication. The goal of this replication step is to load the replicative helicase on the single-stranded DNA generated close to the initiator/DNA complex. In bacteria, two main initiation pathways have been described so far. The PriA-dependent pathway is the best characterized in B. subtilis. It is required to restart DNA synthesis at disassembled replication forks. Dissection of the pathway by genetic and biochemical means revealed the following scheme. First, PriA binds DNA forks carrying single-stranded DNA coated with single-stranded DNA-binding protein and recruits DnaD. The complex then assists the binding of DnaB which recruits DnaI and DnaC. This cascade of reactions yields helicase DnaC loading on the appropriate single-stranded DNA by a ring-assembly mechanism (Bruand et al., 2001, 2005; Marsin et al., 2001; Polard et al., 2002; Velten et al., 2003). The second initiation pathway, the DnaA-dependent pathway, operates at the chromosomal origin of replication. It requires a DnaA/origin interaction as an initial step followed by a cascade of reactions involving DnaB, DnaC, DnaD and DnaI (for reviews, see Lemon et al., 2002; Yoshikawa & Wake, 1993). It is speculated that this cascade is similar to that occurring in the PriA-dependent pathway (Bruand et al., 2005; Velten et al., 2003). It is interesting to note here that DnaD interacts with DnaA and PriA (Bruand et al., 2005; Ishigo-Oka et al., 2001; Marsin et al., 2001). This interaction might thus play a key role in bringing DnaB, DnaI and DnaC to the initiator/DNA complexes.
The factors required for pBS72 initiation of replication are two plasmid elements, the origin and the RepA protein, and four host-encoded replication factors, namely DnaD, DnaB, DnaI and DnaC. It is, however, surprisingly independent of the initiator proteins DnaA and PriA (so far, only ori from R6K, R1, pLS32, pAM1 and RSF1010 have been shown to be DnaA-independent; Bernander et al., 1991; Espinosa et al., 2000; Hassan et al., 1997; Kelley & Bastia, 1992; Scherzinger et al., 1991; Tang et al., 1989). This suggests that the recruitment of the DnaD-driven cascade to the RepA/origin complex might differ from that occurring at the DnaA/oriC and PriA/DNA complexes. A simple model would be that the single-stranded DNA generated by RepA at the plasmid origin is used as a landing pad for DnaD which has a single-stranded DNA-binding activity (Bruand et al., 2005; Marsin et al., 2001). In support of this hypothesis, it was previously shown that the DnaC helicase can be loaded on long stretches of single-stranded DNA via a weakly efficient DnaA- and PriA-independent pathway that requires DnaD, DnaB and DnaI (the DnaD pathway) (Bruand et al., 2005; Marsin et al., 2001). However, similar to all initiator/DNA complexes studied in detail so far [for reviews, see del Solar et al., 1998; Helinski et al., 1996), RepA must not generate a single-stranded region large enough to activate the DnaD pathway at the origin. Additional factors should come into play to compensate for this. Interestingly, several studies show that plasmid initiators interact with host-encoded replication proteins for initiating DNA synthesis [RepA of pSC101 (Datta et al., 1999; Sharma et al., 2001), TrfA of RK2 (Pacek et al., 2001) and 
of R6K (Abhyankar et al., 2004; Lu et al., 1998; Ratnakar et al., 1996); for a review, see Konieczny, 2003]. Thus, it is conceivable that RepA interacts with replication factors to stimulate the DnaD pathway. One possible interacting protein would be DnaD itself as this enzyme is already known to interact with the initiators DnaA and PriA (Bruand et al., 2005; Ishigo-Oka et al., 2001; Marsin et al., 2001). However, RepA carries a region homologous to domain I of DnaA-type proteins which, in the E. coli enzyme, strongly binds the replicative helicase and is required for stabilizing it in the prepriming complex at oriC (Felczak et al., 2005; Seitz et al., 2000; Sutton et al., 1998). Hence, instead of, or in addition to, interacting with DnaD, RepA may bind to DnaC, acting in place of DnaA to efficiently recruit and stabilize the helicase in the plasmid prepriming complex, increasing the efficiency of the DnaD pathway.
The final aspect of pBS72 replication uncovered by this analysis is that once the prepriming complex has formed, the DnaG primase is recruited for synthesizing RNA primers, opening a route for the assembly of the replication machinery. Actually, this machinery is indistinguishable from that acting on the chromosome and the plasmid pAM1 [Bruand et al., 1995; Ceglowski et al., 1993; Dervyn et al., 2001 (for reviews, see Lemon et al., 2002; Winston, 1982; Yoshikawa & Wake, 1993)]. It includes the two DNA polymerases PolC and DnaE, the replication machinery organizer DnaX and the processivity factor DnaN. According to studies carried out in Streptococcus pyogenes (Bruck & O'Donnell, 2000), it should also contain the and ' proteins. The lack of mutations in these genes has precluded their study in this work. Hence, three different genomes (pBS72, pAM1 and the B. subtilis chromosome) are replicated by related, and possibly identical, machinery requiring two different type C polymerases, PolC and DnaE. The substantial differences in the mechanism of DnaC loading in the three genomes (the DnaA pathway at the chromosomal origin, the PriA pathway in pAM1 and putatively the DnaD pathway in pBS72) suggest that this machinery operates in any Cairns replication in B. subtilis and presumably also in related bacteria of the Bacillus/Clostridium and Thermotogales groups.
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ACKNOWLEDGEMENTS |
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dnaA and isogenic strains, Alexandro Galizzi for strain PB1669, Patrice Polard for the priA strain, and N. C. Brown and G. E. Wright for making available HB-EMAU. Nathalie Galleron is thanked for her excellent technical assistance in DNA sequencing and Emmanuelle Le Chatelier, Danielle Canceill and David Lane for stimulating discussions and critical reading of the manuscript. The work was supported, in part, by a research fund from INTAS (INTAS97-1464). |
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Received 17 November 2005;
revised 20 December 2005;
accepted 17 January 2006.
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