Anatomy of the Replication Origin of Plasmid ColE2-P9
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《细菌学杂志》
Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano 390-8621,Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
ABSTRACT
The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.
INTRODUCTION
DNA replication initiates at a specific chromosomal region called the replication origin. The DNA sequence elements required for replication origin activity have been extensively defined and characterized (30). Typically, they consist of a recognition element for the initiator protein and an AT-rich region. In the process of initiation of replication, the initiator protein binds to the origin and induces local unwinding of the duplex DNA. Subsequent events are the loading of a DNA helicase onto the unwound region followed by recruitment of additional factors to initiate DNA synthesis. In the Escherichia coli replication system, the ATP-bound DnaA proteins bind to the 9-mer DnaA boxes in oriC, and then the DnaB helicase is loaded onto unwound 13-mer AT-rich sequences, followed by loading of proteins required for initiation of DNA synthesis, including the DnaG primase (30).
The plasmid ColE2-P9 (ColE2) is a circular duplex DNA molecule of about 7 kb (18) and is present at about 10 to 15 copies per host chromosome (4, 21). The plasmid specifies an initiator protein (Rep protein, 35 kDa), which is the only plasmid-specified trans-acting factor essential for its replication (21, 26, 60). In addition, plasmid replication requires the host DNA polymerase I (28, 50) along with other host factors. The minimal cis-acting region (origin) (Fig. 1) required for plasmid replication consists of only 32 bp, where replication initiates (23, 60). The ColE2 Rep protein specifically binds to the ColE2 origin as revealed by a filter binding assay (26). The cloned origin inhibits replication of autonomously replicating plasmids (IncB incompatibility activity) due to competition for the Rep proteins (51). In vitro ColE2 DNA replication starts at a unique site in the origin region and proceeds unidirectionally (23). The ColE2 Rep protein synthesizes a unique primer RNA (ppApGpA) for initiation of leading-strand DNA synthesis by DNA polymerase I at the origin (53, 54). This results in formation of a D-loop structure, where various host factors are loaded to continue DNA synthesis by DNA polymerase III holoenzyme. The plasmid ColE2-P9 belongs to a large family of very closely related plasmids (ColE2-related plasmids), of which 17 members are currently known (8, 58, 59). The nucleotide sequences of the replicon regions of 11 representative plasmids have been determined (19, 60). All these plasmids share the identical priming mechanism mediated by the plasmid-specified Rep protein. Among them there are four different specificity groups for interaction of the Rep proteins with the origins (19). Based on comparisons of the amino acid sequences of the Rep proteins and the nucleotide sequences of the origins, we have proposed that the specificity of interaction of the Rep proteins with the origins among these four groups of plasmids might be determined by combinations of the amino acid sequences in the three regions in the C-terminal portions of the Rep proteins and the nucleotide sequences at the three sites in the origins. By using chimeric rep genes and chimeric origins along with site-directed mutagenesis, we showed that the specificity between plasmids ColE2 and ColE3-CA38 (ColE3) is determined by combinations of the two sites in the origins with an insertion or deletion of a single base pair (Fig. 1) and corresponding combinations of amino acid sequences in the two regions in the C-terminal portions of the Rep proteins (42). For one of the regions in the Rep protein, the deletion/insertion of a nine-amino-acid sequence is the key factor for determination of the specificity. For the other region in the Rep protein, the specificity seems to be determined by a few amino acids.
By using various derivatives of the ColE2 origin with mutations including deletions and a complete set of single-base-pair substitutions, we analyzed the structural and functional organizations of the ColE2 origin and showed that the region may be divided into at least three subregions. We discuss the functional roles of the three subregions.
MATERIALS AND METHODS
Bacterial strains and plasmids. The Escherichia coli K-12 strains used have been described elsewhere (19, 21, 23, 26). BL21(DE3) (Novagen) was used for production of the Rep protein. The plasmids used have been described elsewhere (21, 26, 42, 55) except for those described below.
Construction of derivatives of plasmid ColE2 origins with deletions and single-base-pair substitutions. Plasmids carrying derivatives of the ColE2 origin (Fig. 1) with deletions from the left (designated dl) and the right (designated dr) were constructed as described at http://science.shinshu-u.ac.jp/bios/PhysDev/tatei/supple/jb0993-05.pdf. Briefly, deletion mutants were generated by digestion with an exonuclease from either side, and single-base-pair mutants were made by using mutagenic oligonucleotides.
Construction of pET21a-E2Rep. pET21a(+) (Novagen) was digested with BglII, filled with T4 DNA polymerase, and self-ligated to construct pET21a(+)BglII. Plasmid pBE243RI-II (a gift from M. Shinohara), containing an EcoRI site just ahead of the rep gene, was constructed from pBE243 (42) by using oligonucleotide EcoRI-ATG. The 5.4-kb EcoRI-XhoI (filled with T4 DNA polymerase) fragment of pET21a(+)BglII was ligated with the 0.9-kb EcoRI-BglII (filled with T4 DNA polymerase) fragment of pBE243RI-II to construct pET21a-E2Rep.
Oligonucleotides used. Oligonucleotides E2-UP and E2-DN have been described previously (60). Other oligonucleotides used in this study are listed at http://science.shinshu-u.ac.jp/bios/PhysDev/tatei/supple/jb0993-05.pdf.
Other materials. The media used have been described previously (19, 21, 23). Chemicals, antibiotics, and enzymes, including some kits, were from commercial sources. Oligonucleotides used for site-directed mutagenesis were supplied by Pharmacia.
Measurement of the replication activity of mutant origins. Plasmid pMB9 was introduced into AG1 recA1 cells carrying the test plasmids (pTI20dr, pTI21dl, and pTI20 point mutant ori series), and then the bacteria were cultured without selection for the test plasmids for 60 generations. One hundred independent colonies were tested for the presence or absence of the test plasmids to determine the retention frequency. The test plasmids were introduced into the bacteria with or without pMB9 to determine the relative transformation frequency. The relative transformation frequency of the test plasmids was estimated by using plasmid-free bacteria.
Incompatibility test and measurement of plasmid copy number. The test plasmids (pBR322dl, pBR322dr, pTI51dl, pTI51dr, and pBlue point mutant ori series) were introduced into AG1 recA1 cells carrying pEC22. The bacteria were cultured without selection for pEC22 for 60 generations. One hundred independent colonies were tested for the presence or absence of pEC22 to determine the retention frequency. The plasmid pEC22 or pTI184 (21), a derivative of pACYC184 (7), was introduced into the bacteria with or without each of the test plasmids. The relative transformation frequency of pEC22 was normalized by using plasmid-free bacteria and pTI184. Measurement of plasmid copy number was performed as described previously (43).
Purification of Rep protein. BL21(DE3) cells harboring pET21a-E2Rep were grown in 100 ml of L broth containing ampicillin (200 μg/ml) at 37°C to 2 x 108 cells/ml. Rep protein production was induced by treatment with 400 μM isopropyl-thio--galactoside for 2 h. Cells were harvested and resuspended in 1 ml of buffer A (50 mM NaH2PO4, 1 M NaCl, 0.5% polyoxyethylene sorbitan monolaurate, pH 8.0) containing 20 mM imidazole and 1 mM phenylmethylsulfonyl fluoride and were stored frozen at –80°C until use. All manipulations were done on ice unless otherwise stated. Frozen cells were thawed, incubated for 30 min with egg white lysozyme (1 mg/ml), sonicated, and clarified by centrifugation at 12,000 x g for 30 min at 4°C. The supernatant was mixed with 200 μl Ni2+-nitrilotriacetic acid resin (50% slurry in buffer A). The resin was washed with buffer A containing 60 mM imidazole and eluted with buffer A containing 250 mM imidazole. The eluate was adjusted to final concentrations of 1 M Tris-HCl, pH 8.0, 0.01% bovine serum albumin, 1 mM dithiothreitol, 0.85 M NaCl, and 50% glycerol and stored at –20°C. The purity of the Rep protein was about 90%, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue.
Gel mobility shift assay. Binding reactions were performed in a final volume of 20 μl containing 400 mM Tris-HCl, pH 7.5, 200 mM KCl, 20 mM MgSO4, 0.4 mM EDTA, 20% ethylene glycol, 5 nM 32P-end-labeled DNA fragment, and an appropriate amount of the Rep protein. Salmon sperm DNA (0.5 μg) was added as a nonspecific competitor. After incubation at 25°C for 30 min, the reaction mixtures were separated on 6% native polyacrylamide gels (10 by 10 cm) at 300 V (constant voltage) for 30 min in 0.5x Tris-borate-EDTA buffer at 4°C. Visualization of the DNA fragments was performed with a Fuji BAS 1500 phosphorimager.
RESULTS AND DISCUSSION
Fine mapping of the boundaries of a functional origin. We have previously shown that the 32-bp region of plasmid ColE2 (positions 7 to 38; Fig. 1) is capable of functioning as the specific replication origin in the presence of the ColE2 Rep protein (60). To define the 5' (left) and 3' (right) ends of the origin more precisely, we constructed various deletion derivatives of the ColE2 origin (Fig. 2) with deletions from the left (dl55, dl4, dl3, dl1, dl2, dl53, and dl5) and from the right (dr27, dr55, dr61, dr62, dr56, dr4, 2-dA, dr8, and dr31) as described at http://science.shinshu-u.ac.jp/bios/PhysDev/tatei/supple/jb0993-05.pdf. The replication activity of mutant origins was measured by a method using plasmid pMB9 as described previously (60). Plasmid ColE1 or its derivatives are readily lost from growing bacteria when the host bacteria also carry pMB9 (17, 56). When plasmid ColE1 carries another unrelated replicon (the ColE2 replicon in this study), it can be maintained by bacteria with coexisting pMB9, as it is capable of replication by use of the second replicon. When the ColE1-ColE2 composite plasmid carries a partially defective ColE2 origin, establishment of the ColE1-ColE2 composite plasmid introduced into the cells already loaded with pMB9 is more difficult than its retention by the cells carrying the composite plasmid into which pMB9 is introduced. Therefore, the relative transformation frequency is the better measure for detecting a relatively mild effect of mutations on the replication activity of the ColE2 origin, whereas the retention frequency is the better measure for detecting a relatively severe effect. When pMB9 was introduced into bacteria carrying derivatives of plasmid ColE1 with cloned mutant ColE2 origins and the ColE2 rep gene (Table 1), those with dl55, dl3, dl1, dl2, dl53, dr27, dr55, dr61, and dr62 were maintained with pMB9, indicating that these mutant origins are active as the functional origin of replication. Those carrying the rest of the mutant origins were all readily lost, indicating that they are inactive. On the other hand, when plasmids with mutant ColE2 origins were introduced into bacteria carrying pMB9 (Table 1), the relative transformation frequencies for dl55, dl3, dr27, dr55, and dr61 were as high as that for the intact ColE2 origin, whereas the frequencies were a little lower for dl1 and dr62 and considerably lower for dl2 and dl53. These results showed that the 5' end of the minimum origin required for replication is at position 8 and that the sequence up to position 5 is required for the full activity. These results also showed that the 3' end of the minimum origin required for replication is located at position 34 or 35 and that the region up to position 36 is required for the full activity.
The Rep protein binding activities of these mutant origins, including dl4 (Table 1), were estimated by their IncB incompatibility activities, namely, inhibition of ColE2 replication due to titration of the Rep protein by binding to the cloned origin (51). For that purpose, the mutant origins were cloned on pBR322 and pNT51, a derivative of ColE1 with a copy number of 200 per host chromosome (56). Establishment of the ColE2 replicon in the cells carrying pNT51 derivatives with the partially defective mutant ColE2 origin is more difficult than that in the cells carrying pBR322 derivatives. Therefore, the relative transformation frequency of the cells carrying pNT51 derivatives is the better measure for detecting a relatively severe effect of mutations on the Rep protein binding activity of the mutant ColE2 origin, whereas that of the cells carrying pBR322 derivatives is the better measure for detecting a relatively mild effect. When an autonomously replicating plasmid ColE2 (pEC22) was introduced into bacteria carrying derivatives of pNT51 with the cloned mutant origins (Table 1), no transformants were obtained for dl55, dl3, dr27, dr55, and dr56, while the relative transformation frequencies were very low for dr4 and 2-dA, low for dl1, and very high for dl2, dl53, dl5, dr8, and dr31, compared with bacteria carrying the pNT51 vector plasmid. On the other hand, when pEC22 was introduced into bacteria carrying pBR322 derivatives (Table 1), no transformants were obtained for dl55, dr27, and dr55, while the relative transformation frequencies were low for dl4, dl3, dr56, dr4, and 2-dA. These results showed that the mutant origins dl55, dr27, and dr55 are fully active, whereas the mutant origin dr56 is a little less active, the mutant origins dl4 and dl3 are less active, and the mutant origins dl1, dr4, and 2-dA are much less active. The IncB incompatibility activities of dl2, dl53, dl5, dr8, and dr31 were undetectable by this method. Although the IncB incompatibility activities of dl2 and dl53 were undetectable, they are active as the functional origin to support plasmid replication, as described above, indicating that they must be capable of interacting with the Rep protein. In contrast, the mutant origins dr56, dr4, and 2-dA retain a significant level of IncB incompatibility activity and yet are inactive for replication. Binding of the Rep protein to dl5 was not detected by DNase I footprint analysis even in the presence of a large excess of the protein (34), indicating that the left end of the minimum origin required for the Rep protein binding is at position 8.
Effects of single-base-pair substitutions on origin activity. Deletion analyses of the ColE2 origin described above showed that the origin is divided into at least two subregions. To locate the boundaries of the functional ColE2 origin more precisely and to examine the significance of each position within the minimum origin for DNA replication and Rep protein binding, we introduced three single-base-pair substitutions at each position of positions 1 to 37 (Fig. 1) as described at http://science.shinshu-u.ac.jp/bios/PhysDev/tatei/supple/jb0993-05.pdf.
The replication activities of these mutant origins carrying single-base-pair substitutions were measured as described in the previous section (Table 2 and Fig. 3). All the mutants with mutations in the region from position 1 to 14 retained the replication activity. From position 1 to 4, all the mutations had no effect on the replication activity. From position 5 to 10, some of the mutations slightly decreased the replication activity. These results together with the results of deletion analyses (Table 1) indicated involvement of positions 5 to 8 in the replication activity. One of the mutations at position 11 showed significantly lower activity, while the other two showed no effects. From position 12 to 14, some of the mutations slightly decreased the replication activity. In the region from position 15 to 37, most of the mutations significantly decreased the activity, except for positions 18, 21, 23, 30, 31, 36, and 37, where mutations had only little or no effect. Mutations at many positions decreased the replication activity below the detectable level, indicating the importance of these positions for replication activity. The results with mutations at position 34 are consistent with those of the deletion analysis using dr56 (Table 1). The results shown here indicated that the 3' end of the minimum origin for replication is located at position 35.
The effects of the single-base-pair substitutions on Rep protein binding were also estimated by measuring the IncB incompatibility activity as described in the previous section (Table 2 and Fig. 3). The effects of mutations at each position on IncB incompatibility activity were strikingly different from those on DNA replication. From position 1 to 4, mutations had little or no effect. In the region from position 5 to 26, all the mutations except for those at positions 13 and 21 greatly affected the IncB incompatibility activity. The G/C base pair at position 6 in the site of the ColE2 origin (Fig. 1) is the specificity determinant in binding of the Rep proteins to the origins in plasmids ColE2 and ColE3 (42). The specificity determinant in the ColE2 Rep protein is region B near the C-terminal end. The amino acid sequence around region B shows significant homology with the DNA recognition helices of various DNA binding proteins (47, 48) and some homology with the helix-turn-helix motif further extends to the N-terminal proximal region of region B (19). Therefore, the region from position 5 to 12 (site a; Fig. 3) containing the G/C base pair atposition 6 might be involved in the specific recognition for Rep protein binding. This region (subregion I) seems to be separated from the adjacent region by the nucleotide at position 13. In the region from position 5 to 15, some mutations decreased the IncB incompatibility activity below the detectable level, although all these mutant origins retained the replication activity as described above (Table 2). By using gel mobility shift assay, we detected weak but significant Rep protein binding activity of these mutant origins, and the results with ori 10A/T and ori 11G/C are shown in Fig. 4.
In the region from position 14 to 26 (subregion II; Fig. 3), many mutations severely decreased the IncB incompatibility activity and the replication activity. Another DNA binding domain of the Rep protein might recognize the region from position 14 to 20 (site b; Fig. 3). The region from position 22 to 26 might be the third recognition element (site c; Fig. 3) for Rep protein binding. Some mutations in subregion II decreased the IncB incompatibility activity nearly below the detectable level and still retained the replication activity. By gel mobility shift assay, we detected weak but significant Rep protein binding activity of these mutant origins, and the result with ori 20G/C is shown in Fig. 4.
The A/T base pair in the ColE3 origin inserted at the corresponding position between positions 21 and 22 at the site of the ColE2 origin (Fig. 1) is the specificity determinant in binding of the Rep proteins to the origins in the plasmids ColE2 and ColE3, and it was proposed to be a spacer (42). Mutations at position 21 hardly affected the IncB incompatibility activity and replication activity. Therefore, the T/A base pair at position 21 in the ColE2 origin and the corresponding base pair in the ColE3 origin (along with the inserted A/T base pair) might be a spacer between the two recognition elements (sites b and c). Even the region from position 21 to 23 could be a spacer. Mutations at position 22, however, severely affected the IncB incompatibility activity and the replication activity. These mutations might cause a conformational change of the origin DNA structure, and the proper interaction of these recognition elements with the Rep protein might be very important for the following step, probably primer RNA synthesis.
In the region from position 27 to 35 (subregion III; Fig. 3) mutations had only little effect on the IncB incompatibility activity. At positions 36 and 37, mutations had no effect on the activity. The results with mutations at positions 28 to 30 are apparently a little inconsistent with those of deletion analyses with dr4 and 2-dA (Table 2). Multiple substitutions in the sequence to the right of positions 28 and 29 in dr4 and 2-dA, respectively, might have affected the IncB incompatibility activity. Even though the region from position 32 to 35 is not covered with the Rep protein (34), this region is required for the replication and might interact with a host factor required for replication. Initiation of plasmid ColE2 replication requires DNA polymerase I, which could be a candidate. DNA polymerase I is known to bind to highly repetitive bacterial DNA sequences called the palindromic unit (PU) in a sequence-specific manner (15). There are two regions in the ColE2 origin that show high homology with a part of the PU (Fig. 5), although both are outside subregion III. These sequences might still act to recruit DNA polymerase I for efficient utilization of the primer RNA. We are currently trying to detect specific interaction of DNA polymerase I with the ColE2 origin.
In the ColE2 origin there are two perfect direct repeats of 5'-CAGATAA-3' (Fig. 1). Repeated sequences are quite common in the origin sequences of various DNA replication systems (10). In every case the sequence is the recognition element for the initiator protein. The present study clearly showed that these two sequences in the ColE2 origin are functionally distinct. The 3' half of the direct repeat on the right is not covered by the Rep protein as revealed by DNase I footprint analysis (34).
Properties of autonomously replicating plasmids carrying mutant origins. To examine the properties of the mutant origins with deletions from the left (dl1, dl2, dl3, and dl53) in more detail, we constructed autonomously replicating plasmids carrying the mutant origins and rep gene (Table 3). The relative transformation frequencies of these plasmids carrying mutant origins dl1, dl2, and dl53 were considerably lower than those of plasmids with the intact origin. Plasmids with mutant origins dl2 and dl53 were lost from about 2% of host bacteria after 40 generations and from 10% after 100 generations without selection for resistance to the antibiotics. Plasmids with the intact origin were stably maintained by host bacteria at least for 100 generations. We then examined the copy numbers of these autonomously replicating plasmids in exponentially growing bacteria (Table 3). Unexpectedly, the plasmids carrying mutant origins dl1, dl2, and dl53, with much lower relative transformation frequency, were maintained by host bacteria with significantly higher copy numbers than those of other plasmids. The plasmid with dl3 also exhibited some increase in the copy number. The properties of the mutant origin dl55 were quite similar to those of the intact origin.
We further examined the copy numbers of autonomously replicating plasmids carrying mutant origins with single-base-pair substitutions (Table 4). For the mutant origins with Rep protein binding activity decreased below the detectable level and with (near-) normal replication activity, the copy numbers of the autonomously replicating plasmids were 1.6 to 2.7 times higher than that of the wild-type origin. For some mutant origins with the binding activity decreased below the detectable level and with the replication activity reduced severely, the relative copy numbers were still 1.0 to 2.1. For the mutant origins in which mutations had a significant effect on the Rep protein binding activity, the relative copy numbers of the plasmids with mutant origins in subregion I were 1.3 to 2.5 and those of plasmids with mutant origins in subregion II were 0.7 to 1.1. For those mutant origins in which mutations had a little or no effect on both the Rep protein binding activity and replication activity, the relative copy numbers were 0.7 to 1.7.
These results showed that some of the mutant origins of subregion I with Rep protein binding activity below the detectable level may initiate replication more efficiently than the wild-type origin. We suggest that efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. This possibility has been previously suggested by the study of the chimeric origins and Rep proteins between plasmids ColE2 and ColE3 (42).
Models for binding of the Rep protein to the origin. Based upon the results describe above, we discuss possible models. If we assume that the Rep protein binds to the origin as a monomer, the helix-turn-helix motif in the C-terminal region of the Rep protein (19) recognizes and binds to site a (Fig. 3), and the putative second and third DNA binding domains specifically bind to sites b and c separated by the spacer. The N-terminal domain of the Rep protein might recognize and bind to the region around site c to synthesize the primer RNA. The spacer might be important for the proper positioning of the N-terminal domain on the template DNA strand. If we assume that the ColE2 Rep protein binds to the origin as a dimer, there could be two alternative models. In either model, two distinct binding domains in the C-terminal half of one of the Rep proteins in the dimer recognizes sites a and b and binds to the origin DNA, and the spacer might be important for the proper positioning of the second Rep protein. Binding of the second Rep protein to the origin is facilitated by protein-protein interaction. In one model, the third DNA recognition domain of the second Rep protein in the dimer recognizes site c and catalyzes the primer RNA synthesis. In the other model, either of the two recognition domains of the second Rep protein in the dimer interacts with site c, which is only weakly homologous to site a or b. Although the Rep proteins form stable dimers even in the presence of 1 M NaCl (35), so far we do not know whether the Rep protein binds to the origin as a monomer or a dimer (or as an oligomer).
ColE2-related plasmids in gram-negative and gram-positive bacteria. For the past 10 years or so, growing numbers of plasmids whose putative Rep proteins are related to the ColE2-P9 Rep protein have been described (Fig. 6 and 7) (1, 2, 3, 5, 6, 9, 11, 12, 13, 14, 16, 19, 20, 22, 25, 27, 29, 31, 32, 33, 36, 37, 38, 39, 40, 41, 44, 45, 46, 49, 52, 57, 61, 62). For the ColE2-related plasmids, such as plasmids ColE2-P9, ColE3-CA38, ColE5-099, and ColE2-CA42, the origin sequences where the Rep proteins bind in a plasmid-specific manner and initiate plasmid replication have been identified (19, 23, 53, 60). These origin sequences showed good homology with each other, and the sequences around the primer sequence are almost identical (Fig. 6). For only some of the remaining plasmids have the origin sequences been proposed. For the rest of the plasmids, we searched for candidates based on the sequence homology with the origins of the ColE2-related plasmids (Fig. 6 and 7). The putative origin sequences of pEMCJH03 proposed here are very similar to the ColE2-P9 origin sequence. The putative replication origins of pFAJ2600, namely, ori1, ori2, and ori3 (11) and ori4 (32), have been mentioned. We propose here three other sequences (ori5, ori6, and ori7) as better candidates based on the higher similarity with the origins of the ColE2-related plasmids, which partly overlap ori1, ori3, and ori2, respectively, in the opposite orientation. For pXZ10142, ori1 and ori2 (11) and ori3 (32) have been proposed, and we propose here another candidate (ori4). ori3 and ori4 are apparently better candidates than ori1 and ori2. Similarly ori2 of pAL5000 seems to be a better candidate than ori1. Both the RepB binding H site and L site are located close to ori1 and ori2 (45).
The sequence 5'-ATCAGATA-3' is found in the putative origins of most of the plasmids from the gram-negative bacteria (Fig. 6). Closely related sequences are found in pUB6060 (ori1), pTik12, pCPP519, pTT8 (ori1), pXAC33, and pHE1. The sequence 5'-ATCAGATA-3' or similar sequences are also found in many of the plasmids from the gram-positive bacteria (Fig. 7). This sequence is located across the boundary of subregions II and III of the ColE2-P9 origin (Fig. 3), contains the primer sequence, and is very important for in vivo replication as described above. The DnaG primase of E. coli has been shown to interact with the DNA sequences upstream and downstream of the primer start position (24). The catalytic domains of the Rep proteins of the ColE2-related plasmids for primer synthesis might interact with the 5'-TATCTGAT-3' sequence on the template strand. It can be speculated that all these plasmids (Fig. 6 and 7) share a common mechanism of initiation of DNA replication, that is, the primer RNA synthesis by the Rep proteins. It is noteworthy that these plasmids are widespread in gram-positive and -negative bacteria and form a large plasmid family.
ACKNOWLEDGMENTS
We are grateful to H. Ogawa and T. Ogawa for helpful discussions and continuing encouragement. We thank T. Horii, A. Shinohara, M. Shinohara, H. Kubo, and Y. Itoh for discussion and A. Ohshima Desaki, M. Shinohara, K. Matsumoto, J. Itoh, T. Tanigaki, and Y. Suzuki for materials used. We also thank G. Minote for radioisotope experiments in the Research Center for Human and Environmental Sciences of Shinshu University.
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.
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ABSTRACT
The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.
INTRODUCTION
DNA replication initiates at a specific chromosomal region called the replication origin. The DNA sequence elements required for replication origin activity have been extensively defined and characterized (30). Typically, they consist of a recognition element for the initiator protein and an AT-rich region. In the process of initiation of replication, the initiator protein binds to the origin and induces local unwinding of the duplex DNA. Subsequent events are the loading of a DNA helicase onto the unwound region followed by recruitment of additional factors to initiate DNA synthesis. In the Escherichia coli replication system, the ATP-bound DnaA proteins bind to the 9-mer DnaA boxes in oriC, and then the DnaB helicase is loaded onto unwound 13-mer AT-rich sequences, followed by loading of proteins required for initiation of DNA synthesis, including the DnaG primase (30).
The plasmid ColE2-P9 (ColE2) is a circular duplex DNA molecule of about 7 kb (18) and is present at about 10 to 15 copies per host chromosome (4, 21). The plasmid specifies an initiator protein (Rep protein, 35 kDa), which is the only plasmid-specified trans-acting factor essential for its replication (21, 26, 60). In addition, plasmid replication requires the host DNA polymerase I (28, 50) along with other host factors. The minimal cis-acting region (origin) (Fig. 1) required for plasmid replication consists of only 32 bp, where replication initiates (23, 60). The ColE2 Rep protein specifically binds to the ColE2 origin as revealed by a filter binding assay (26). The cloned origin inhibits replication of autonomously replicating plasmids (IncB incompatibility activity) due to competition for the Rep proteins (51). In vitro ColE2 DNA replication starts at a unique site in the origin region and proceeds unidirectionally (23). The ColE2 Rep protein synthesizes a unique primer RNA (ppApGpA) for initiation of leading-strand DNA synthesis by DNA polymerase I at the origin (53, 54). This results in formation of a D-loop structure, where various host factors are loaded to continue DNA synthesis by DNA polymerase III holoenzyme. The plasmid ColE2-P9 belongs to a large family of very closely related plasmids (ColE2-related plasmids), of which 17 members are currently known (8, 58, 59). The nucleotide sequences of the replicon regions of 11 representative plasmids have been determined (19, 60). All these plasmids share the identical priming mechanism mediated by the plasmid-specified Rep protein. Among them there are four different specificity groups for interaction of the Rep proteins with the origins (19). Based on comparisons of the amino acid sequences of the Rep proteins and the nucleotide sequences of the origins, we have proposed that the specificity of interaction of the Rep proteins with the origins among these four groups of plasmids might be determined by combinations of the amino acid sequences in the three regions in the C-terminal portions of the Rep proteins and the nucleotide sequences at the three sites in the origins. By using chimeric rep genes and chimeric origins along with site-directed mutagenesis, we showed that the specificity between plasmids ColE2 and ColE3-CA38 (ColE3) is determined by combinations of the two sites in the origins with an insertion or deletion of a single base pair (Fig. 1) and corresponding combinations of amino acid sequences in the two regions in the C-terminal portions of the Rep proteins (42). For one of the regions in the Rep protein, the deletion/insertion of a nine-amino-acid sequence is the key factor for determination of the specificity. For the other region in the Rep protein, the specificity seems to be determined by a few amino acids.
By using various derivatives of the ColE2 origin with mutations including deletions and a complete set of single-base-pair substitutions, we analyzed the structural and functional organizations of the ColE2 origin and showed that the region may be divided into at least three subregions. We discuss the functional roles of the three subregions.
MATERIALS AND METHODS
Bacterial strains and plasmids. The Escherichia coli K-12 strains used have been described elsewhere (19, 21, 23, 26). BL21(DE3) (Novagen) was used for production of the Rep protein. The plasmids used have been described elsewhere (21, 26, 42, 55) except for those described below.
Construction of derivatives of plasmid ColE2 origins with deletions and single-base-pair substitutions. Plasmids carrying derivatives of the ColE2 origin (Fig. 1) with deletions from the left (designated dl) and the right (designated dr) were constructed as described at http://science.shinshu-u.ac.jp/bios/PhysDev/tatei/supple/jb0993-05.pdf. Briefly, deletion mutants were generated by digestion with an exonuclease from either side, and single-base-pair mutants were made by using mutagenic oligonucleotides.
Construction of pET21a-E2Rep. pET21a(+) (Novagen) was digested with BglII, filled with T4 DNA polymerase, and self-ligated to construct pET21a(+)BglII. Plasmid pBE243RI-II (a gift from M. Shinohara), containing an EcoRI site just ahead of the rep gene, was constructed from pBE243 (42) by using oligonucleotide EcoRI-ATG. The 5.4-kb EcoRI-XhoI (filled with T4 DNA polymerase) fragment of pET21a(+)BglII was ligated with the 0.9-kb EcoRI-BglII (filled with T4 DNA polymerase) fragment of pBE243RI-II to construct pET21a-E2Rep.
Oligonucleotides used. Oligonucleotides E2-UP and E2-DN have been described previously (60). Other oligonucleotides used in this study are listed at http://science.shinshu-u.ac.jp/bios/PhysDev/tatei/supple/jb0993-05.pdf.
Other materials. The media used have been described previously (19, 21, 23). Chemicals, antibiotics, and enzymes, including some kits, were from commercial sources. Oligonucleotides used for site-directed mutagenesis were supplied by Pharmacia.
Measurement of the replication activity of mutant origins. Plasmid pMB9 was introduced into AG1 recA1 cells carrying the test plasmids (pTI20dr, pTI21dl, and pTI20 point mutant ori series), and then the bacteria were cultured without selection for the test plasmids for 60 generations. One hundred independent colonies were tested for the presence or absence of the test plasmids to determine the retention frequency. The test plasmids were introduced into the bacteria with or without pMB9 to determine the relative transformation frequency. The relative transformation frequency of the test plasmids was estimated by using plasmid-free bacteria.
Incompatibility test and measurement of plasmid copy number. The test plasmids (pBR322dl, pBR322dr, pTI51dl, pTI51dr, and pBlue point mutant ori series) were introduced into AG1 recA1 cells carrying pEC22. The bacteria were cultured without selection for pEC22 for 60 generations. One hundred independent colonies were tested for the presence or absence of pEC22 to determine the retention frequency. The plasmid pEC22 or pTI184 (21), a derivative of pACYC184 (7), was introduced into the bacteria with or without each of the test plasmids. The relative transformation frequency of pEC22 was normalized by using plasmid-free bacteria and pTI184. Measurement of plasmid copy number was performed as described previously (43).
Purification of Rep protein. BL21(DE3) cells harboring pET21a-E2Rep were grown in 100 ml of L broth containing ampicillin (200 μg/ml) at 37°C to 2 x 108 cells/ml. Rep protein production was induced by treatment with 400 μM isopropyl-thio--galactoside for 2 h. Cells were harvested and resuspended in 1 ml of buffer A (50 mM NaH2PO4, 1 M NaCl, 0.5% polyoxyethylene sorbitan monolaurate, pH 8.0) containing 20 mM imidazole and 1 mM phenylmethylsulfonyl fluoride and were stored frozen at –80°C until use. All manipulations were done on ice unless otherwise stated. Frozen cells were thawed, incubated for 30 min with egg white lysozyme (1 mg/ml), sonicated, and clarified by centrifugation at 12,000 x g for 30 min at 4°C. The supernatant was mixed with 200 μl Ni2+-nitrilotriacetic acid resin (50% slurry in buffer A). The resin was washed with buffer A containing 60 mM imidazole and eluted with buffer A containing 250 mM imidazole. The eluate was adjusted to final concentrations of 1 M Tris-HCl, pH 8.0, 0.01% bovine serum albumin, 1 mM dithiothreitol, 0.85 M NaCl, and 50% glycerol and stored at –20°C. The purity of the Rep protein was about 90%, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue.
Gel mobility shift assay. Binding reactions were performed in a final volume of 20 μl containing 400 mM Tris-HCl, pH 7.5, 200 mM KCl, 20 mM MgSO4, 0.4 mM EDTA, 20% ethylene glycol, 5 nM 32P-end-labeled DNA fragment, and an appropriate amount of the Rep protein. Salmon sperm DNA (0.5 μg) was added as a nonspecific competitor. After incubation at 25°C for 30 min, the reaction mixtures were separated on 6% native polyacrylamide gels (10 by 10 cm) at 300 V (constant voltage) for 30 min in 0.5x Tris-borate-EDTA buffer at 4°C. Visualization of the DNA fragments was performed with a Fuji BAS 1500 phosphorimager.
RESULTS AND DISCUSSION
Fine mapping of the boundaries of a functional origin. We have previously shown that the 32-bp region of plasmid ColE2 (positions 7 to 38; Fig. 1) is capable of functioning as the specific replication origin in the presence of the ColE2 Rep protein (60). To define the 5' (left) and 3' (right) ends of the origin more precisely, we constructed various deletion derivatives of the ColE2 origin (Fig. 2) with deletions from the left (dl55, dl4, dl3, dl1, dl2, dl53, and dl5) and from the right (dr27, dr55, dr61, dr62, dr56, dr4, 2-dA, dr8, and dr31) as described at http://science.shinshu-u.ac.jp/bios/PhysDev/tatei/supple/jb0993-05.pdf. The replication activity of mutant origins was measured by a method using plasmid pMB9 as described previously (60). Plasmid ColE1 or its derivatives are readily lost from growing bacteria when the host bacteria also carry pMB9 (17, 56). When plasmid ColE1 carries another unrelated replicon (the ColE2 replicon in this study), it can be maintained by bacteria with coexisting pMB9, as it is capable of replication by use of the second replicon. When the ColE1-ColE2 composite plasmid carries a partially defective ColE2 origin, establishment of the ColE1-ColE2 composite plasmid introduced into the cells already loaded with pMB9 is more difficult than its retention by the cells carrying the composite plasmid into which pMB9 is introduced. Therefore, the relative transformation frequency is the better measure for detecting a relatively mild effect of mutations on the replication activity of the ColE2 origin, whereas the retention frequency is the better measure for detecting a relatively severe effect. When pMB9 was introduced into bacteria carrying derivatives of plasmid ColE1 with cloned mutant ColE2 origins and the ColE2 rep gene (Table 1), those with dl55, dl3, dl1, dl2, dl53, dr27, dr55, dr61, and dr62 were maintained with pMB9, indicating that these mutant origins are active as the functional origin of replication. Those carrying the rest of the mutant origins were all readily lost, indicating that they are inactive. On the other hand, when plasmids with mutant ColE2 origins were introduced into bacteria carrying pMB9 (Table 1), the relative transformation frequencies for dl55, dl3, dr27, dr55, and dr61 were as high as that for the intact ColE2 origin, whereas the frequencies were a little lower for dl1 and dr62 and considerably lower for dl2 and dl53. These results showed that the 5' end of the minimum origin required for replication is at position 8 and that the sequence up to position 5 is required for the full activity. These results also showed that the 3' end of the minimum origin required for replication is located at position 34 or 35 and that the region up to position 36 is required for the full activity.
The Rep protein binding activities of these mutant origins, including dl4 (Table 1), were estimated by their IncB incompatibility activities, namely, inhibition of ColE2 replication due to titration of the Rep protein by binding to the cloned origin (51). For that purpose, the mutant origins were cloned on pBR322 and pNT51, a derivative of ColE1 with a copy number of 200 per host chromosome (56). Establishment of the ColE2 replicon in the cells carrying pNT51 derivatives with the partially defective mutant ColE2 origin is more difficult than that in the cells carrying pBR322 derivatives. Therefore, the relative transformation frequency of the cells carrying pNT51 derivatives is the better measure for detecting a relatively severe effect of mutations on the Rep protein binding activity of the mutant ColE2 origin, whereas that of the cells carrying pBR322 derivatives is the better measure for detecting a relatively mild effect. When an autonomously replicating plasmid ColE2 (pEC22) was introduced into bacteria carrying derivatives of pNT51 with the cloned mutant origins (Table 1), no transformants were obtained for dl55, dl3, dr27, dr55, and dr56, while the relative transformation frequencies were very low for dr4 and 2-dA, low for dl1, and very high for dl2, dl53, dl5, dr8, and dr31, compared with bacteria carrying the pNT51 vector plasmid. On the other hand, when pEC22 was introduced into bacteria carrying pBR322 derivatives (Table 1), no transformants were obtained for dl55, dr27, and dr55, while the relative transformation frequencies were low for dl4, dl3, dr56, dr4, and 2-dA. These results showed that the mutant origins dl55, dr27, and dr55 are fully active, whereas the mutant origin dr56 is a little less active, the mutant origins dl4 and dl3 are less active, and the mutant origins dl1, dr4, and 2-dA are much less active. The IncB incompatibility activities of dl2, dl53, dl5, dr8, and dr31 were undetectable by this method. Although the IncB incompatibility activities of dl2 and dl53 were undetectable, they are active as the functional origin to support plasmid replication, as described above, indicating that they must be capable of interacting with the Rep protein. In contrast, the mutant origins dr56, dr4, and 2-dA retain a significant level of IncB incompatibility activity and yet are inactive for replication. Binding of the Rep protein to dl5 was not detected by DNase I footprint analysis even in the presence of a large excess of the protein (34), indicating that the left end of the minimum origin required for the Rep protein binding is at position 8.
Effects of single-base-pair substitutions on origin activity. Deletion analyses of the ColE2 origin described above showed that the origin is divided into at least two subregions. To locate the boundaries of the functional ColE2 origin more precisely and to examine the significance of each position within the minimum origin for DNA replication and Rep protein binding, we introduced three single-base-pair substitutions at each position of positions 1 to 37 (Fig. 1) as described at http://science.shinshu-u.ac.jp/bios/PhysDev/tatei/supple/jb0993-05.pdf.
The replication activities of these mutant origins carrying single-base-pair substitutions were measured as described in the previous section (Table 2 and Fig. 3). All the mutants with mutations in the region from position 1 to 14 retained the replication activity. From position 1 to 4, all the mutations had no effect on the replication activity. From position 5 to 10, some of the mutations slightly decreased the replication activity. These results together with the results of deletion analyses (Table 1) indicated involvement of positions 5 to 8 in the replication activity. One of the mutations at position 11 showed significantly lower activity, while the other two showed no effects. From position 12 to 14, some of the mutations slightly decreased the replication activity. In the region from position 15 to 37, most of the mutations significantly decreased the activity, except for positions 18, 21, 23, 30, 31, 36, and 37, where mutations had only little or no effect. Mutations at many positions decreased the replication activity below the detectable level, indicating the importance of these positions for replication activity. The results with mutations at position 34 are consistent with those of the deletion analysis using dr56 (Table 1). The results shown here indicated that the 3' end of the minimum origin for replication is located at position 35.
The effects of the single-base-pair substitutions on Rep protein binding were also estimated by measuring the IncB incompatibility activity as described in the previous section (Table 2 and Fig. 3). The effects of mutations at each position on IncB incompatibility activity were strikingly different from those on DNA replication. From position 1 to 4, mutations had little or no effect. In the region from position 5 to 26, all the mutations except for those at positions 13 and 21 greatly affected the IncB incompatibility activity. The G/C base pair at position 6 in the site of the ColE2 origin (Fig. 1) is the specificity determinant in binding of the Rep proteins to the origins in plasmids ColE2 and ColE3 (42). The specificity determinant in the ColE2 Rep protein is region B near the C-terminal end. The amino acid sequence around region B shows significant homology with the DNA recognition helices of various DNA binding proteins (47, 48) and some homology with the helix-turn-helix motif further extends to the N-terminal proximal region of region B (19). Therefore, the region from position 5 to 12 (site a; Fig. 3) containing the G/C base pair atposition 6 might be involved in the specific recognition for Rep protein binding. This region (subregion I) seems to be separated from the adjacent region by the nucleotide at position 13. In the region from position 5 to 15, some mutations decreased the IncB incompatibility activity below the detectable level, although all these mutant origins retained the replication activity as described above (Table 2). By using gel mobility shift assay, we detected weak but significant Rep protein binding activity of these mutant origins, and the results with ori 10A/T and ori 11G/C are shown in Fig. 4.
In the region from position 14 to 26 (subregion II; Fig. 3), many mutations severely decreased the IncB incompatibility activity and the replication activity. Another DNA binding domain of the Rep protein might recognize the region from position 14 to 20 (site b; Fig. 3). The region from position 22 to 26 might be the third recognition element (site c; Fig. 3) for Rep protein binding. Some mutations in subregion II decreased the IncB incompatibility activity nearly below the detectable level and still retained the replication activity. By gel mobility shift assay, we detected weak but significant Rep protein binding activity of these mutant origins, and the result with ori 20G/C is shown in Fig. 4.
The A/T base pair in the ColE3 origin inserted at the corresponding position between positions 21 and 22 at the site of the ColE2 origin (Fig. 1) is the specificity determinant in binding of the Rep proteins to the origins in the plasmids ColE2 and ColE3, and it was proposed to be a spacer (42). Mutations at position 21 hardly affected the IncB incompatibility activity and replication activity. Therefore, the T/A base pair at position 21 in the ColE2 origin and the corresponding base pair in the ColE3 origin (along with the inserted A/T base pair) might be a spacer between the two recognition elements (sites b and c). Even the region from position 21 to 23 could be a spacer. Mutations at position 22, however, severely affected the IncB incompatibility activity and the replication activity. These mutations might cause a conformational change of the origin DNA structure, and the proper interaction of these recognition elements with the Rep protein might be very important for the following step, probably primer RNA synthesis.
In the region from position 27 to 35 (subregion III; Fig. 3) mutations had only little effect on the IncB incompatibility activity. At positions 36 and 37, mutations had no effect on the activity. The results with mutations at positions 28 to 30 are apparently a little inconsistent with those of deletion analyses with dr4 and 2-dA (Table 2). Multiple substitutions in the sequence to the right of positions 28 and 29 in dr4 and 2-dA, respectively, might have affected the IncB incompatibility activity. Even though the region from position 32 to 35 is not covered with the Rep protein (34), this region is required for the replication and might interact with a host factor required for replication. Initiation of plasmid ColE2 replication requires DNA polymerase I, which could be a candidate. DNA polymerase I is known to bind to highly repetitive bacterial DNA sequences called the palindromic unit (PU) in a sequence-specific manner (15). There are two regions in the ColE2 origin that show high homology with a part of the PU (Fig. 5), although both are outside subregion III. These sequences might still act to recruit DNA polymerase I for efficient utilization of the primer RNA. We are currently trying to detect specific interaction of DNA polymerase I with the ColE2 origin.
In the ColE2 origin there are two perfect direct repeats of 5'-CAGATAA-3' (Fig. 1). Repeated sequences are quite common in the origin sequences of various DNA replication systems (10). In every case the sequence is the recognition element for the initiator protein. The present study clearly showed that these two sequences in the ColE2 origin are functionally distinct. The 3' half of the direct repeat on the right is not covered by the Rep protein as revealed by DNase I footprint analysis (34).
Properties of autonomously replicating plasmids carrying mutant origins. To examine the properties of the mutant origins with deletions from the left (dl1, dl2, dl3, and dl53) in more detail, we constructed autonomously replicating plasmids carrying the mutant origins and rep gene (Table 3). The relative transformation frequencies of these plasmids carrying mutant origins dl1, dl2, and dl53 were considerably lower than those of plasmids with the intact origin. Plasmids with mutant origins dl2 and dl53 were lost from about 2% of host bacteria after 40 generations and from 10% after 100 generations without selection for resistance to the antibiotics. Plasmids with the intact origin were stably maintained by host bacteria at least for 100 generations. We then examined the copy numbers of these autonomously replicating plasmids in exponentially growing bacteria (Table 3). Unexpectedly, the plasmids carrying mutant origins dl1, dl2, and dl53, with much lower relative transformation frequency, were maintained by host bacteria with significantly higher copy numbers than those of other plasmids. The plasmid with dl3 also exhibited some increase in the copy number. The properties of the mutant origin dl55 were quite similar to those of the intact origin.
We further examined the copy numbers of autonomously replicating plasmids carrying mutant origins with single-base-pair substitutions (Table 4). For the mutant origins with Rep protein binding activity decreased below the detectable level and with (near-) normal replication activity, the copy numbers of the autonomously replicating plasmids were 1.6 to 2.7 times higher than that of the wild-type origin. For some mutant origins with the binding activity decreased below the detectable level and with the replication activity reduced severely, the relative copy numbers were still 1.0 to 2.1. For the mutant origins in which mutations had a significant effect on the Rep protein binding activity, the relative copy numbers of the plasmids with mutant origins in subregion I were 1.3 to 2.5 and those of plasmids with mutant origins in subregion II were 0.7 to 1.1. For those mutant origins in which mutations had a little or no effect on both the Rep protein binding activity and replication activity, the relative copy numbers were 0.7 to 1.7.
These results showed that some of the mutant origins of subregion I with Rep protein binding activity below the detectable level may initiate replication more efficiently than the wild-type origin. We suggest that efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. This possibility has been previously suggested by the study of the chimeric origins and Rep proteins between plasmids ColE2 and ColE3 (42).
Models for binding of the Rep protein to the origin. Based upon the results describe above, we discuss possible models. If we assume that the Rep protein binds to the origin as a monomer, the helix-turn-helix motif in the C-terminal region of the Rep protein (19) recognizes and binds to site a (Fig. 3), and the putative second and third DNA binding domains specifically bind to sites b and c separated by the spacer. The N-terminal domain of the Rep protein might recognize and bind to the region around site c to synthesize the primer RNA. The spacer might be important for the proper positioning of the N-terminal domain on the template DNA strand. If we assume that the ColE2 Rep protein binds to the origin as a dimer, there could be two alternative models. In either model, two distinct binding domains in the C-terminal half of one of the Rep proteins in the dimer recognizes sites a and b and binds to the origin DNA, and the spacer might be important for the proper positioning of the second Rep protein. Binding of the second Rep protein to the origin is facilitated by protein-protein interaction. In one model, the third DNA recognition domain of the second Rep protein in the dimer recognizes site c and catalyzes the primer RNA synthesis. In the other model, either of the two recognition domains of the second Rep protein in the dimer interacts with site c, which is only weakly homologous to site a or b. Although the Rep proteins form stable dimers even in the presence of 1 M NaCl (35), so far we do not know whether the Rep protein binds to the origin as a monomer or a dimer (or as an oligomer).
ColE2-related plasmids in gram-negative and gram-positive bacteria. For the past 10 years or so, growing numbers of plasmids whose putative Rep proteins are related to the ColE2-P9 Rep protein have been described (Fig. 6 and 7) (1, 2, 3, 5, 6, 9, 11, 12, 13, 14, 16, 19, 20, 22, 25, 27, 29, 31, 32, 33, 36, 37, 38, 39, 40, 41, 44, 45, 46, 49, 52, 57, 61, 62). For the ColE2-related plasmids, such as plasmids ColE2-P9, ColE3-CA38, ColE5-099, and ColE2-CA42, the origin sequences where the Rep proteins bind in a plasmid-specific manner and initiate plasmid replication have been identified (19, 23, 53, 60). These origin sequences showed good homology with each other, and the sequences around the primer sequence are almost identical (Fig. 6). For only some of the remaining plasmids have the origin sequences been proposed. For the rest of the plasmids, we searched for candidates based on the sequence homology with the origins of the ColE2-related plasmids (Fig. 6 and 7). The putative origin sequences of pEMCJH03 proposed here are very similar to the ColE2-P9 origin sequence. The putative replication origins of pFAJ2600, namely, ori1, ori2, and ori3 (11) and ori4 (32), have been mentioned. We propose here three other sequences (ori5, ori6, and ori7) as better candidates based on the higher similarity with the origins of the ColE2-related plasmids, which partly overlap ori1, ori3, and ori2, respectively, in the opposite orientation. For pXZ10142, ori1 and ori2 (11) and ori3 (32) have been proposed, and we propose here another candidate (ori4). ori3 and ori4 are apparently better candidates than ori1 and ori2. Similarly ori2 of pAL5000 seems to be a better candidate than ori1. Both the RepB binding H site and L site are located close to ori1 and ori2 (45).
The sequence 5'-ATCAGATA-3' is found in the putative origins of most of the plasmids from the gram-negative bacteria (Fig. 6). Closely related sequences are found in pUB6060 (ori1), pTik12, pCPP519, pTT8 (ori1), pXAC33, and pHE1. The sequence 5'-ATCAGATA-3' or similar sequences are also found in many of the plasmids from the gram-positive bacteria (Fig. 7). This sequence is located across the boundary of subregions II and III of the ColE2-P9 origin (Fig. 3), contains the primer sequence, and is very important for in vivo replication as described above. The DnaG primase of E. coli has been shown to interact with the DNA sequences upstream and downstream of the primer start position (24). The catalytic domains of the Rep proteins of the ColE2-related plasmids for primer synthesis might interact with the 5'-TATCTGAT-3' sequence on the template strand. It can be speculated that all these plasmids (Fig. 6 and 7) share a common mechanism of initiation of DNA replication, that is, the primer RNA synthesis by the Rep proteins. It is noteworthy that these plasmids are widespread in gram-positive and -negative bacteria and form a large plasmid family.
ACKNOWLEDGMENTS
We are grateful to H. Ogawa and T. Ogawa for helpful discussions and continuing encouragement. We thank T. Horii, A. Shinohara, M. Shinohara, H. Kubo, and Y. Itoh for discussion and A. Ohshima Desaki, M. Shinohara, K. Matsumoto, J. Itoh, T. Tanigaki, and Y. Suzuki for materials used. We also thank G. Minote for radioisotope experiments in the Research Center for Human and Environmental Sciences of Shinshu University.
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.
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