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SeqA, the Escherichia coli origin sequestration protein, can regulate the replication of the pBR322 plasmid
Plasmid 65 (2011) 15–19
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Plasmid journal homepage: www.elsevier.com/locate/yplas
SeqA, the Escherichia coli origin sequestration protein, can regulate the replication of the pBR322 plasmid Daghfous Douraid ⇑, Landoulsi Ahmed Laboratoire de Biochimie et de Biologie Moléculaire, Faculté des sciences de Bizerte, 7021 Zarzouna, Tunisia
a r t i c l e
i n f o
Article history: Received 21 October 2009 Accepted 17 September 2010 Available online 25 September 2010 Commuicated by Grzegorz Wegrzyn Keywords: SeqA pBR322 Sequestration oriC oripBR322 Membrane
a b s t r a c t The pBR322 plasmid origin replication and oriC show similar responses to adenine methylation. Both are subject to sequestration by membrane fractions. In fact, like the host origin oriC, the RNA II promoter region of pBR322 is regulated by methylation of three GATC adenine methylation sites. The SeqA gene product acts in the negative control of oriC by sequestration. We suggest that the role of SeqA protein in sequestration is similar to oriC region DNA. Hence, SeqA recognize the methylation state of the pBR322RNA II promoter region by direct DNA binding in vitro. Using the pOC42 plasmid, we show that SeqA binds exclusively to the hemimethylated form of the replication origin of the pBR322 plasmid. In addition, we suggested that the SeqA protein could modulate periodically the initiation of replication of the pBR322 plasmid. The later could be fixed by its origin sequence, on a hemimethylated state, during the initiation of the replication. Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction Escherichia coli cell cycle is maintained trough strict controls and checkpoints coordinating replication and partition of the bacterial chromosome. It is clear that a mechanism exists to prevent premature re-initiation until the proper time in the cell cycle is reached (Skarstad et al., 1986). Several factors have been proposed to play a role in the sequestration of newly replicated origins to prevent premature re-initiation during eclipse period. The methylation state of chromosomal origin has been implicated both in the binding of oriC to the cell membrane and in the prevention of premature re-initiation at newly replicated origins (Landoulsi et al., 1990). In E. coli, adenine residues within chromosomal GATC sequences are methylated in a reaction catalyzed by Dam methylase (Herman and Mordrich, 1982). Since the methylation reactions occur after replication, adenine residues within GATC sequences in the newly synthesized strand remain unmethylated for ⇑ Corresponding author. Fax.: +216 72 590 566. E-mail address: [email protected] (D. Douraid).
a period of time. The oriC region contains a high density of GATC sites, and adenine residues methylation at several sites is delayed significantly relative to the methylation time at GATC sites elsewhere in the chromosome (Ogden et al., 1988; Campbell and Kleckner, 1990). As a result, the oriC region of newly replicated chromosomes remains hemimethylated for about 30–40% of the cell cycle (Von Freiesleben et al., 1994). Hemimethylated oriC evidence does not serve as an effective template for initiation of replication in vivo came from the finding of Russell and Zinder (Russell and Zinder, 1987) that a fully methylated oriC plasmid was unable to replicate in a dam- host. Under these conditions, the first round replication in the dam mutant gives rise to hemimethylated oriC that is never fully methylated because of the defect in Dam methylase. It was suggested that the hemimethylated origin was refractory to re-initiation, thereby explaining the defect in plasmid replication (Ogden et al., 1988) then showed that hemimethylated oriC DNA binds preferentially to membrane fractions in vitro. Landoulsi and colleagues (1990) demonstrated that the association of hemimethylated oriC with the membrane plays a direct role in preventing
0147-619X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2010.09.003
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D. Douraid, L. Ahmed / Plasmid 65 (2011) 15–19
initiation of replication in vitro. These results support the idea that the membrane association of hemimethylated oriC plays a key role in the postreplication block of re-initiation that occurs during the normal cell cycle (Daghfous et al., 2006). SeqA is a protein that is required for both the membrane binding of hemimethylated oriC in vitro and the refractory period to initiation that follows replication of the origin region in dam+ cells (Lu et al., 1994; Slater et al., 1995; Boye et al., 1996). Indeed, SeqA plays a role in sequestering the newly replicated origin by making it inaccessible to the replication machinery, and suggests that this may involve a SeqA-mediated oriC-membrane interaction (Waldminghaus and Skarstad, 2009; Daghfous et al., 2009). The replication origin of the pBR322 plasmid, oripBR322 (Sutcliffe, 1978) and oriC show parallel responses to adenine methylation (Russell and Zinder, 1987) and both are subject to sequestration (Malki et al., 1991). In fact, the same transformation studies (Messer et al., 1980; Smith et al., 1985; Russell and Zinder, 1987) demonstrate that the replication of the ColE1-type plasmid pBR322 is also inhibited by hemimethylation although the effect is less dramatic than seen with oriC plasmids. A promoter located 555 bp upstream of the replication origin furnishes primers for the initiation of ColE1 DNA replication and contains three GATC sites, two of which are conserved amongst all ColE1-derivatives plasmid (Chan and Williams, 1995). Patnaik and colleagues (1990) have shown that disruption of these sites by oligonucleotide-directed mutagenesis relieves hemimethylation-mediated inhibition of DNA replication in vivo. In addition, Malki et al. (1991) have shown that specific sequestration of the hemimethylated RNA II promoter region by the outer cell membrane results in a reduction in initiation frequency at the oripBR322 DNA sequence. In this report, using the pOC42 plasmid, we show that SeqA binds exclusively to the hemimethylated form of oripBR322. We suggest also that the SeqA protein could modulate periodically the pBR322 initiation of replication by fixing its origin sequence, on a hemimethylated state. 2. Experimental procedures 2.1. Bacterial strains and plasmids Escherichia coli K12 strains, PA3092 (prototrophic) and GM2199 (dam13::Tn9, thr-1, ara-14, leuB6, tonA31, lacY1, hisG4) (Marinus et al., 1983) were used as a source of methylated and nonmethylated pOC42 plasmids, respectively. The SeqA protein was isolated as described previously (Taghbalout et al., 2000). FII replication extracts were isolated from E. coli strain WM1177 (dnaA46ts, polAI, endoI, thy, F) (Ohmori et al., 1984). 2.2. Preparation of plasmid DNA The 6 Kb plasmid pOC42 contains the entire oriC fragment cloned into PstI site of pBR322 (Messer et al., 1985). Methylated DNA and unmethylated DNA was obtained from
the prototrophic strain PA3092, respectively (Marinus et al., 1983) and the dam13 mutant GM2199 by the alkaline extraction procedure (Birnboim and Doly, 1979), followed by 1 M NaCl gradients and caesium chloride/ethidium bromide equilibrium centrifugation (Maniatis et al., 1982). Hemimethylated pOC42 DNA was prepared by re-annealing methylated and unmethylated DNA strands, respectively, after cutting with the restriction endonucleases SmaI and EcoRV as described previously (Landoulsi et al., 1989). 32 P end-labled restriction fragments were prepared by a HinfI–EcoRI double digest of pOC42, followed by end labelling with Klenow fragment and [a-32P] dATP. This method and subsequent purification were carried out according to Maniatis and colleagues (1982). 2.3. Purification of the SeqA protein SeqA protein was purified according to the Taghbalout and colleagues technique (Taghbalout et al., 2000) with modifications. The SeqA protein was over-expressed in the YM901 E. coli strain harbouring the pMY161 plasmid. This plasmid produces a SeqA protein fused at its C-terminal to eight Histidine residues. The his-tagged SeqA is able to function in vivo. The YM901 strain was cultured at 30 °C on LB-medium supplemented with ampicillin (100 lg/ml) and chloramphenicol (30 lg/ml) and the synthesis of SeqA protein was induced by IPTG. The cells were grown with IPTG for 1–2 generations before harvesting at 4000 rpm. The cells pellet were re-suspended in an equal weight of buffer A (50 mM Tris (pH 7.5); 5 mM EDTA;1 mM DTT). The cells were lysed by freezing and thawing, and the cells lysate was centrifuged in a Beckman 50 Ti rotor (45 min, 45000 rpm, 4 °C). The supernatant was removed and proteins were precipitated with 30% ammonium sulfate. The precipitate was dialysed in buffer A supplemented with 1 M NaCl. The SeqA protein was purified using 5 ml of chelating Sepharose FF (Pharmacia) coupled with nickel ion. Purified SeqA protein was dialysed against dilution buffer (buffer A plus 1 M NaCl). The protein concentration was measured according to the Bradford method (Bradford, 1976). 2.4. Enzyme extracts used for in vitro DNA replication Fraction II enzyme extracts were prepared from E. coli strain WM1177 (dnaA46ts, polAI, endoI, thy, F) (Ohmori et al., 1984) according to Fuller et al. (1981). Particular care was taken to ensure that bacteria were harvested at the same optical density and treated to obtain identical extracts. Protein concentrations, measured by the Bradford method (Bradford, 1976), were in the range of 80–90 mg/ml. 2.5. In vitro oriC/oripBR322 plasmid replication assays In vitro DNA replication was carried out according to Fuller et al. (1981) with modifications. Assays were in a volume of 50 ll and unlabelled deoxyribonucleotides were at 50 lM. Radioactive [methyl-3H]-TTP (40 Ci/mmole) was at 0.75 lCi and 4 ll of enzyme extract was added per reaction. The pOC42 oriC and oripBR322 plasmid DNA (8 lg/ml) was incubated at 0 °C for 5 min in 50 ll of reaction mixture
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(25 mM HEPES–KOH (pH 7.6), 0.1 mM EDTA, 2 mM DTT, 5 mM magnesium-acetate, 4 mM ATP, 40 mM creatine phosphate, 10 lg creatine kinase, 0.05 mg/ml of BSA, 6% PEG 6000, 0.5 mM NTPs, 0.1 mM dNTPs (except [3H]-dTTP, 0.75 lCi/mmol) with or without the SeqA protein (0–10 lg). Contamination of SeqA in this fraction was negligible (less than 2 ng SeqA/100 lg of fraction II, as judged by a sensitive chemiluminescence western blotting analysis (d’Alençon et al., 1999). When oriC dependent DNA replication was assayed, the mixture was incubated for 20 min at 30 °C with 5 lg of DnaA and 240 lg of fraction II obtained from E. coli strain WM1177. In the case of pBR322 dependent DNA replication, the final concentration of the assay mixture was adjusted to 100 mM KCl and the DnaA protein fraction was omitted. The radioactivity in acid insoluble precipitate was counted to measure DNA synthesis.
(-/-) B
FT
(+/+) B
FT
(+/-) B
FT oriC (1272 pb)
oripBR322 (517 pb)
2.6. Nitrocellulose binding reactions 0.02 lg of radioactive Hinf I–EcoRI restricted pOC42 DNA was mixed with 5 lg of cold calf thymus DNA and then incubated with 4 lg of SeqA at 37 °C for 15 min in 50 ll of binding buffer (Fuller et al., 1984). Calf thymus DNA was omitted from the DNA protein binding assays. Reaction mixtures were then filtered through nitrocellulose membrane filters, washed at room temperature with binding buffer. After the elution, purification, and concentration of the DNA in both bound and flow-through fractions, they were subjected to 4% polyacrylamide gel electrophoresis (Laemmli, 1970). 3. Results 3.1. SeqA protein possesses specific affinity for hemimethylated DNA fragments containing oriC and the RNA II promoter region of pBR322 Membrane filter binding studies have shown that hemimethylated oriC DNA fragments are bound specifically by the SeqA protein (Landoulsi et al., 1990). In order to test the possibility that a similar mechanism also operates to inhibit hemimethylated pBR3222 DNA replication, first we examined the SeqA protein affinity for DNA restriction fragments from the joint replicon pOC42. The latter contains in addition to the entire pBR322 molecule, the PstI oriC fragment of the chromosome (Landoulsi et al., 1990). Thus, we prepared the fully methylated, hemimethylated and unmethylated state of the plasmid pOC42 and 32P end-labeled restriction endonuclease digests of these DNA’s were incubated in the presence of the SeqA protein and then filtered on Nitrocellulose membrane filters. As found previously, the DNA fragment of 1272 bp, corresponding to the hemimethylated oriC sequence is specifically retained on the filters (Landoulsi et al., 1990) (Fig. 1). In addition, another DNA fragment of 517 bp, corresponding to a region upstream of the pBR322 origin, is observed. This fragment contains the RNA II primer promoter region, where a cluster of three GATC sequences, has been shown to mediate hemimethylated inhibition of pBR322 DNA replication (Landoulsi
Fig. 1. 32P end-labled HinfI–EcoRI restriction fragments of methylated (+/+), unmethylated ( / ), and hemimethylated (+/ ) pOC42 DNA were preincubated with 4 lg of SeqA protein and filtered on nitrocellulose membrane filters. DNA-bound (B) and flow-through fractions (FT) from the filters were subjected to polyacrylamide gel electrophoresis. The 517 bp RNA II promoter region of pBR322 DNA fragment and the 1272 bp oriC DNA fragment are indicated by arrows.
et al., 1990). Methylated and unmethylated DNA sequences of oriC and oripBR322 were weakly bound (Fig. 1). 3.2. SeqA Inhibits initiations both at oriC and the origin of pBR322 Under specific conditions, enhancing initiation at oriC on pOC42, DNA synthesis is preferentially inhibited by SeqA protein when hemimethylated is used (Fig. 2A). This inhibition is much less severe when methylated or unmethylated pOC42 templates are used (Fig. 2A). In Fig. 2(B), we show the results of a similar experiment but realized this time when nearly all the initiations occur at the pBR322 origin on pOC42 (conditions in experimental procedures). As well as initiations at oriC, initiation at the pBR322 origin can occur in vitro when the template is hemimethylated. However, the addition of the SeqA protein to the assays results in a specific inhibition of pBR322 replication on the hemimethylated pOC42 plasmid. In addition, a comparison of Fig. 2(A) and (B) demonstrates that more SeqA protein is required to inhibit initiations at the pBR322 replication origin. This is consistent with the differential affinities of the SeqA protein for oriC and the RNA II promoter DNA fragments. 3.3. Omitting of the SeqA protein relieves hemimethylation We have re-induced the in vitro replication of the oripBR322 hemimethylated DNA template and we have
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Relative activity of DNA synthesis(%)
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(+/+)
80 60 40
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20 0
0
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SeqA (µg)
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80 60 40 20 0
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SeqA (µg)
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Relative activity of DNA synthesis (%)
(B)
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Relative activity of DNA synthesis(%)
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D. Douraid, L. Ahmed / Plasmid 65 (2011) 15–19
Fig. 3. Effect of various concentrations of SeqA on DNA synthesis from the hemimethylated oripBR322 plasmid DNA in the presence of 4 ll of antiSeqA antibody. Solid triangles (N), hemimethylated oripBR322 DNA; Solid squares (j), hemimethylated oripBR322 DNA supplemented with antiSeqA;
(+/+)
100 80 60 40
(+/-)
20 0
0
1
2
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SeqA (µg) Fig. 2. Effect of SeqA on oriC and oripBR322 plasmid replication in vitro: (A) Inhibition of oriC plasmid DNA replication by various concentrations of SeqA. Standard conditions (5 lg of protein DnaA and without KCl) for in vitro replication of the oriC plasmid pOC42 were used. In the absence of SeqA, the amount of DNA synthesized was 176 pmol with hemimethylated template DNA and 272 pmol with fully methylated template DNA. (s), fully oriC methylated DNA (+/+); (D), hemimethylated oriC DNA (+/ ). (B) Inhibition of oripBR322 plasmid DNA replication by various concentrations of SeqA. The DnaA protein was substituted by 100 mM KCl. In the absence of SeqA, the amount of DNA synthesized was 292 pmoles with hemimethylated template DNA and 276 pmoles with fully methylated template DNA. (d) fully oripBR322 methylated DNA (+/ +); (N) hemimethylated oripBR322 DNA (+/ ).
examined the effect of the addition of the SeqA-antibody to the replication mixture. The addition of the anti-SeqA antibody during the in vitro replication, at the oripBR322 region, blocks specifically the effect of the SeqA protein (Fig. 3). As a result, in this condition, the amount of radioactivity labeled desoxynucleotides incorporated into newly synthesized DNA, was measured. This amount is similar to the amount of radioactivity obtained with the replication starting from an oripBR322 methylated DNA state (Fig. 3). These findings confirm that the inhibition of the initiation of the replication at oripBR322 sequence requires a direct interaction between the SeqA protein and the hemimethylated RNA II promoter region of pBR322.
4. Discussion In this study, we show that hemimethylation-mediated inhibition of pBR322 replication relies on the same mechanism previously described for oriC, i.e., response to adenine methylation (Russell and Zinder, 1987) and the sequestration of the hemimethylated origin region away from initiation by the cell membrane (Malki et al., 1991).
On the basis of earlier studies (Ogden et al., 1988; Landoulsi et al., 1990), Lu et al. (1994) proposed, that hemimethylated oriC binds specifically to a high density membrane fraction in vitro, that SeqA acts negatively on the initiation of replication to sequestrate oriC into the membrane where it is transiently refractory to re-initiation. In vivo, two different effects of SeqA on DNA replication have been distinguished. First, SeqA delays replication initiation without perturbing the precision with which initiation is coupled to cell physiology (Boye et al., 1996). Second, SeqA is required for sequestration of oriC from re-methylation immediately following replication initiation and, in a presumably related effect, for blocking replication of hemimethylated oriC plasmids (Lu et al., 1994; Slater et al., 1995; Taghbalout et al., 2000) and the oriP plasmid (Brendler et al., 1995). As plasmids driven by oripBR322 are also subject to sequestration (Landoulsi et al., 1990; Malki et al., 1991), it is probable that the SeqA protein regulates pBR322 replication of newly synthesized DNA by detecting its hemimethylated state. In vitro replication of pBR322 involves probably sequestration of the RNA II primer promoter away from RNA polymerase by SeqA with a consequential reduction in RNA II primer transcription and initiation frequency at the downstream start site for DNA synthesis. Our results indicate that the SeqA recognition of the RNA II promoter region of pBR322 involves direct DNA binding of the SeqA protein to the hemimethylated origin sequences formed as a result of replication. Thus, SeqA would block initiation directly by preventing binding of essential factors or by interfering with some key event (Waldminghaus and Skarstad, 2009), such as strand opening. This would prevent re-initiation until the unmethylated strands are methylated, thus releasing the SeqA protein. Acknowledgments We are most grateful to Dr. M. Kohiyama for providing bacterial strains, without which this work would not be possible.
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References Birnboim, H.C., Doly, J., 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513– 1523. Boye, E., Stokke, T., Kleckner, N., Skarstad, K., 1996. Coordinating DNA replication initiation with cell growth: differential roles for DnaA and SeqA proteins. Proc. Natl. Acad. Sci. USA 93, 12206–12211. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of micrograme quantities of proteins using the principle of proteindye binding. Anal. Biochem. 72, 248–254. Brendler, T., Abeles, A., Austin, S., 1995. A protein that binds to the P1 origin core and the oriC 13mer region in a methlation-specific fashion is the product of the host seqA gene. EMBO J. 14, 4083–4089. Campbell, J.L., Kleckner, N., 1990. E. coli and the dnaA gene promoter are sequestered from Dam methyltransferase following the passage of chromosomal replication fork. Cell 62, 967–979. Chan, J.W., Williams, K.R., 1995. Single-stranded DNA binding proteins required for DNA replication. Annu. Rev. Biochem. 55, 103–135. Daghfous, D., Chatti, A., Marzouk, B., Landoulsi, A., 2006. Phospholipid changes in seqA and dam mutants of Escherichia coli. CR Biol. 329, 271–276. Daghfous, D., Chatti, A., Hammami, R., Landoulsi, A., 2009. Modelling of the full-length Escherichia coli SeqA protein. In complex with DNA. Pathol. Biol. 57, 61–66. d’Alençon, E., Taghbalout, A., Kern, R., Kohiyama, M., 1999. Replication cycle dependent association of SeqA to the outer membrane fraction of E. coli. Biochimie 81, 841–846. Fuller, R.S., Kaguni, J.M., Kornberg, A., 1981. Enzymatic replication of the origin of the Escherichia coli chromosome. Proc. Natl. Acad. Sci. USA 78, 7370–7374. Fuller, R., Funnell, B., Kornberg, A., 1984. The DnaA complex with the E. coli chromosomal origin and other DnaN sites. Cell 38, 889–900. Herman, G.E., Mordrich, P., 1982. Escherichia coli dam methylase. Physical and catalytical properties of the homogeneous enzyme. J. Biol. Chem. 257, 2605–2612. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Landoulsi, A., Hughes, P., Kern, R., Kohiyama, M., 1989. Dam methylation and the initiation of DNA replication on oriC plasmids. Mol. Gen. Genet. 216, 217–223. Landoulsi, A., Malki, A., Kern, R., Kohiyama, M., Hughes, P., 1990. The E. coli cell surface specifically prevents the initiation of DAN replication at oriC on hemimethylated DNA templates. Cell 63, 1053–1060. Lu, M., Campbell, J.L., Boye, E., Kleckner, N., 1994. SeqA: a negative modulator of replication initiation in E. coli. Cell 77, 413–426.
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Malki, A., Kern, R., Kohiyama, M., Hughes, P., 1991. Inhibition of DNA synthesis at the hemimethylated pBR322 origin of replication by cell membrane. Nucleic Acids. Res. 20, 105–109. Maniatis, T., Fritsh, E.F., Sambrook, J., 1982. Molecular cloning: a laboratory manual. Cold Spring harbor Laboratory Press, Cold Spring Harbor, New York. Marinus, M.G., Carraway, M., Frey, A.Z., Brown, L., Arraj, J.A., 1983. Insertion mutations in the dam gene of Escherichia coli K12. Mol. Gen. Genet. 192, 288–289. Messer, W., Hermann, B., Meijer, M., Hall, S., 1980. Structure of the E. coli replication origin oriC sequence required for maintenance, establishment and bi-directional replication. Symp. Mol. Cell Biol. ICN-UCLA 19, 161–169. Messer, W., Bellekes, U., Lother, H., 1985. Effect of dam methylation on the activity of the E. coli replication origin, oriC. EMBO J. 4, 1327–1332. Ogden, G.B., Pratt, M.J., Schaechter, M., 1988. The replicative origin of the E. coli chromosome binds to cell membranes only when hemimethylated. Cell 54, 127–135. Ohmori, H., Kimura, M., Sakakibara, Y., 1984. Structural analysis of the dnaA and dnaN genes of Escherichia coli. Gene 28, 159–170. Patnaik, P.K., Merlin, S., Plisky, B., 1990. Effect of altering GATC sequences in the plasmid ColE1 primer promoter. J. Bacteriol. 172, 1762–1768. Russell, D.W., Zinder, N.D., 1987. Hemimethylation prevents DNA replication in Escherichia coli. Cell 50, 1071–1079. Skarstad, K., Boye, E., Steen, H.B., 1986. Timing of initiation of chromosome replication in individual E. coli cells. EMBO J. 5, 1711– 1717. Slater, S., Wold, S., Lu, M., Boye, E., Skarstad, K., Kleckner, N., 1995. Escherichia coli SeqA protein binds oriC in two different methylmodulated reactions appropriate to its roles in DNA replication initiation and origin sequestration. Cell 82, 927–936. Smith, D.W., Garland, A.M., Herman, G., Enns, R.E., Baker, T.A., Zyskind, J.W., 1985. Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli. EMBO J. 4, 1319–1326. Sutcliffe, J.G., 1978. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol., 77–90. Taghbalout, A., Landoulsi, A., Kern, R., Yamazoe, M., Hiraga, S., Holland, B., Kohiyama, M., Malki, A., 2000. Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro. Genes Cells 5, 873–884. Von Freiesleben, U., Rasmussen, K.V., Schaechter, M., 1994. SeqA limits DnaA activity in replication from oriC in Escherichia coli. Mol. Microbiol. 14, 763–772. Waldminghaus, T., Skarstad, K., 2009. The Escherichia coli SeqA protein. Plasmid 61, 141–150.
Contents lists available at ScienceDirect
Plasmid journal homepage: www.elsevier.com/locate/yplas
SeqA, the Escherichia coli origin sequestration protein, can regulate the replication of the pBR322 plasmid Daghfous Douraid ⇑, Landoulsi Ahmed Laboratoire de Biochimie et de Biologie Moléculaire, Faculté des sciences de Bizerte, 7021 Zarzouna, Tunisia
a r t i c l e
i n f o
Article history: Received 21 October 2009 Accepted 17 September 2010 Available online 25 September 2010 Commuicated by Grzegorz Wegrzyn Keywords: SeqA pBR322 Sequestration oriC oripBR322 Membrane
a b s t r a c t The pBR322 plasmid origin replication and oriC show similar responses to adenine methylation. Both are subject to sequestration by membrane fractions. In fact, like the host origin oriC, the RNA II promoter region of pBR322 is regulated by methylation of three GATC adenine methylation sites. The SeqA gene product acts in the negative control of oriC by sequestration. We suggest that the role of SeqA protein in sequestration is similar to oriC region DNA. Hence, SeqA recognize the methylation state of the pBR322RNA II promoter region by direct DNA binding in vitro. Using the pOC42 plasmid, we show that SeqA binds exclusively to the hemimethylated form of the replication origin of the pBR322 plasmid. In addition, we suggested that the SeqA protein could modulate periodically the initiation of replication of the pBR322 plasmid. The later could be fixed by its origin sequence, on a hemimethylated state, during the initiation of the replication. Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction Escherichia coli cell cycle is maintained trough strict controls and checkpoints coordinating replication and partition of the bacterial chromosome. It is clear that a mechanism exists to prevent premature re-initiation until the proper time in the cell cycle is reached (Skarstad et al., 1986). Several factors have been proposed to play a role in the sequestration of newly replicated origins to prevent premature re-initiation during eclipse period. The methylation state of chromosomal origin has been implicated both in the binding of oriC to the cell membrane and in the prevention of premature re-initiation at newly replicated origins (Landoulsi et al., 1990). In E. coli, adenine residues within chromosomal GATC sequences are methylated in a reaction catalyzed by Dam methylase (Herman and Mordrich, 1982). Since the methylation reactions occur after replication, adenine residues within GATC sequences in the newly synthesized strand remain unmethylated for ⇑ Corresponding author. Fax.: +216 72 590 566. E-mail address: [email protected] (D. Douraid).
a period of time. The oriC region contains a high density of GATC sites, and adenine residues methylation at several sites is delayed significantly relative to the methylation time at GATC sites elsewhere in the chromosome (Ogden et al., 1988; Campbell and Kleckner, 1990). As a result, the oriC region of newly replicated chromosomes remains hemimethylated for about 30–40% of the cell cycle (Von Freiesleben et al., 1994). Hemimethylated oriC evidence does not serve as an effective template for initiation of replication in vivo came from the finding of Russell and Zinder (Russell and Zinder, 1987) that a fully methylated oriC plasmid was unable to replicate in a dam- host. Under these conditions, the first round replication in the dam mutant gives rise to hemimethylated oriC that is never fully methylated because of the defect in Dam methylase. It was suggested that the hemimethylated origin was refractory to re-initiation, thereby explaining the defect in plasmid replication (Ogden et al., 1988) then showed that hemimethylated oriC DNA binds preferentially to membrane fractions in vitro. Landoulsi and colleagues (1990) demonstrated that the association of hemimethylated oriC with the membrane plays a direct role in preventing
0147-619X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2010.09.003
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D. Douraid, L. Ahmed / Plasmid 65 (2011) 15–19
initiation of replication in vitro. These results support the idea that the membrane association of hemimethylated oriC plays a key role in the postreplication block of re-initiation that occurs during the normal cell cycle (Daghfous et al., 2006). SeqA is a protein that is required for both the membrane binding of hemimethylated oriC in vitro and the refractory period to initiation that follows replication of the origin region in dam+ cells (Lu et al., 1994; Slater et al., 1995; Boye et al., 1996). Indeed, SeqA plays a role in sequestering the newly replicated origin by making it inaccessible to the replication machinery, and suggests that this may involve a SeqA-mediated oriC-membrane interaction (Waldminghaus and Skarstad, 2009; Daghfous et al., 2009). The replication origin of the pBR322 plasmid, oripBR322 (Sutcliffe, 1978) and oriC show parallel responses to adenine methylation (Russell and Zinder, 1987) and both are subject to sequestration (Malki et al., 1991). In fact, the same transformation studies (Messer et al., 1980; Smith et al., 1985; Russell and Zinder, 1987) demonstrate that the replication of the ColE1-type plasmid pBR322 is also inhibited by hemimethylation although the effect is less dramatic than seen with oriC plasmids. A promoter located 555 bp upstream of the replication origin furnishes primers for the initiation of ColE1 DNA replication and contains three GATC sites, two of which are conserved amongst all ColE1-derivatives plasmid (Chan and Williams, 1995). Patnaik and colleagues (1990) have shown that disruption of these sites by oligonucleotide-directed mutagenesis relieves hemimethylation-mediated inhibition of DNA replication in vivo. In addition, Malki et al. (1991) have shown that specific sequestration of the hemimethylated RNA II promoter region by the outer cell membrane results in a reduction in initiation frequency at the oripBR322 DNA sequence. In this report, using the pOC42 plasmid, we show that SeqA binds exclusively to the hemimethylated form of oripBR322. We suggest also that the SeqA protein could modulate periodically the pBR322 initiation of replication by fixing its origin sequence, on a hemimethylated state. 2. Experimental procedures 2.1. Bacterial strains and plasmids Escherichia coli K12 strains, PA3092 (prototrophic) and GM2199 (dam13::Tn9, thr-1, ara-14, leuB6, tonA31, lacY1, hisG4) (Marinus et al., 1983) were used as a source of methylated and nonmethylated pOC42 plasmids, respectively. The SeqA protein was isolated as described previously (Taghbalout et al., 2000). FII replication extracts were isolated from E. coli strain WM1177 (dnaA46ts, polAI, endoI, thy, F) (Ohmori et al., 1984). 2.2. Preparation of plasmid DNA The 6 Kb plasmid pOC42 contains the entire oriC fragment cloned into PstI site of pBR322 (Messer et al., 1985). Methylated DNA and unmethylated DNA was obtained from
the prototrophic strain PA3092, respectively (Marinus et al., 1983) and the dam13 mutant GM2199 by the alkaline extraction procedure (Birnboim and Doly, 1979), followed by 1 M NaCl gradients and caesium chloride/ethidium bromide equilibrium centrifugation (Maniatis et al., 1982). Hemimethylated pOC42 DNA was prepared by re-annealing methylated and unmethylated DNA strands, respectively, after cutting with the restriction endonucleases SmaI and EcoRV as described previously (Landoulsi et al., 1989). 32 P end-labled restriction fragments were prepared by a HinfI–EcoRI double digest of pOC42, followed by end labelling with Klenow fragment and [a-32P] dATP. This method and subsequent purification were carried out according to Maniatis and colleagues (1982). 2.3. Purification of the SeqA protein SeqA protein was purified according to the Taghbalout and colleagues technique (Taghbalout et al., 2000) with modifications. The SeqA protein was over-expressed in the YM901 E. coli strain harbouring the pMY161 plasmid. This plasmid produces a SeqA protein fused at its C-terminal to eight Histidine residues. The his-tagged SeqA is able to function in vivo. The YM901 strain was cultured at 30 °C on LB-medium supplemented with ampicillin (100 lg/ml) and chloramphenicol (30 lg/ml) and the synthesis of SeqA protein was induced by IPTG. The cells were grown with IPTG for 1–2 generations before harvesting at 4000 rpm. The cells pellet were re-suspended in an equal weight of buffer A (50 mM Tris (pH 7.5); 5 mM EDTA;1 mM DTT). The cells were lysed by freezing and thawing, and the cells lysate was centrifuged in a Beckman 50 Ti rotor (45 min, 45000 rpm, 4 °C). The supernatant was removed and proteins were precipitated with 30% ammonium sulfate. The precipitate was dialysed in buffer A supplemented with 1 M NaCl. The SeqA protein was purified using 5 ml of chelating Sepharose FF (Pharmacia) coupled with nickel ion. Purified SeqA protein was dialysed against dilution buffer (buffer A plus 1 M NaCl). The protein concentration was measured according to the Bradford method (Bradford, 1976). 2.4. Enzyme extracts used for in vitro DNA replication Fraction II enzyme extracts were prepared from E. coli strain WM1177 (dnaA46ts, polAI, endoI, thy, F) (Ohmori et al., 1984) according to Fuller et al. (1981). Particular care was taken to ensure that bacteria were harvested at the same optical density and treated to obtain identical extracts. Protein concentrations, measured by the Bradford method (Bradford, 1976), were in the range of 80–90 mg/ml. 2.5. In vitro oriC/oripBR322 plasmid replication assays In vitro DNA replication was carried out according to Fuller et al. (1981) with modifications. Assays were in a volume of 50 ll and unlabelled deoxyribonucleotides were at 50 lM. Radioactive [methyl-3H]-TTP (40 Ci/mmole) was at 0.75 lCi and 4 ll of enzyme extract was added per reaction. The pOC42 oriC and oripBR322 plasmid DNA (8 lg/ml) was incubated at 0 °C for 5 min in 50 ll of reaction mixture
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(25 mM HEPES–KOH (pH 7.6), 0.1 mM EDTA, 2 mM DTT, 5 mM magnesium-acetate, 4 mM ATP, 40 mM creatine phosphate, 10 lg creatine kinase, 0.05 mg/ml of BSA, 6% PEG 6000, 0.5 mM NTPs, 0.1 mM dNTPs (except [3H]-dTTP, 0.75 lCi/mmol) with or without the SeqA protein (0–10 lg). Contamination of SeqA in this fraction was negligible (less than 2 ng SeqA/100 lg of fraction II, as judged by a sensitive chemiluminescence western blotting analysis (d’Alençon et al., 1999). When oriC dependent DNA replication was assayed, the mixture was incubated for 20 min at 30 °C with 5 lg of DnaA and 240 lg of fraction II obtained from E. coli strain WM1177. In the case of pBR322 dependent DNA replication, the final concentration of the assay mixture was adjusted to 100 mM KCl and the DnaA protein fraction was omitted. The radioactivity in acid insoluble precipitate was counted to measure DNA synthesis.
(-/-) B
FT
(+/+) B
FT
(+/-) B
FT oriC (1272 pb)
oripBR322 (517 pb)
2.6. Nitrocellulose binding reactions 0.02 lg of radioactive Hinf I–EcoRI restricted pOC42 DNA was mixed with 5 lg of cold calf thymus DNA and then incubated with 4 lg of SeqA at 37 °C for 15 min in 50 ll of binding buffer (Fuller et al., 1984). Calf thymus DNA was omitted from the DNA protein binding assays. Reaction mixtures were then filtered through nitrocellulose membrane filters, washed at room temperature with binding buffer. After the elution, purification, and concentration of the DNA in both bound and flow-through fractions, they were subjected to 4% polyacrylamide gel electrophoresis (Laemmli, 1970). 3. Results 3.1. SeqA protein possesses specific affinity for hemimethylated DNA fragments containing oriC and the RNA II promoter region of pBR322 Membrane filter binding studies have shown that hemimethylated oriC DNA fragments are bound specifically by the SeqA protein (Landoulsi et al., 1990). In order to test the possibility that a similar mechanism also operates to inhibit hemimethylated pBR3222 DNA replication, first we examined the SeqA protein affinity for DNA restriction fragments from the joint replicon pOC42. The latter contains in addition to the entire pBR322 molecule, the PstI oriC fragment of the chromosome (Landoulsi et al., 1990). Thus, we prepared the fully methylated, hemimethylated and unmethylated state of the plasmid pOC42 and 32P end-labeled restriction endonuclease digests of these DNA’s were incubated in the presence of the SeqA protein and then filtered on Nitrocellulose membrane filters. As found previously, the DNA fragment of 1272 bp, corresponding to the hemimethylated oriC sequence is specifically retained on the filters (Landoulsi et al., 1990) (Fig. 1). In addition, another DNA fragment of 517 bp, corresponding to a region upstream of the pBR322 origin, is observed. This fragment contains the RNA II primer promoter region, where a cluster of three GATC sequences, has been shown to mediate hemimethylated inhibition of pBR322 DNA replication (Landoulsi
Fig. 1. 32P end-labled HinfI–EcoRI restriction fragments of methylated (+/+), unmethylated ( / ), and hemimethylated (+/ ) pOC42 DNA were preincubated with 4 lg of SeqA protein and filtered on nitrocellulose membrane filters. DNA-bound (B) and flow-through fractions (FT) from the filters were subjected to polyacrylamide gel electrophoresis. The 517 bp RNA II promoter region of pBR322 DNA fragment and the 1272 bp oriC DNA fragment are indicated by arrows.
et al., 1990). Methylated and unmethylated DNA sequences of oriC and oripBR322 were weakly bound (Fig. 1). 3.2. SeqA Inhibits initiations both at oriC and the origin of pBR322 Under specific conditions, enhancing initiation at oriC on pOC42, DNA synthesis is preferentially inhibited by SeqA protein when hemimethylated is used (Fig. 2A). This inhibition is much less severe when methylated or unmethylated pOC42 templates are used (Fig. 2A). In Fig. 2(B), we show the results of a similar experiment but realized this time when nearly all the initiations occur at the pBR322 origin on pOC42 (conditions in experimental procedures). As well as initiations at oriC, initiation at the pBR322 origin can occur in vitro when the template is hemimethylated. However, the addition of the SeqA protein to the assays results in a specific inhibition of pBR322 replication on the hemimethylated pOC42 plasmid. In addition, a comparison of Fig. 2(A) and (B) demonstrates that more SeqA protein is required to inhibit initiations at the pBR322 replication origin. This is consistent with the differential affinities of the SeqA protein for oriC and the RNA II promoter DNA fragments. 3.3. Omitting of the SeqA protein relieves hemimethylation We have re-induced the in vitro replication of the oripBR322 hemimethylated DNA template and we have
18
120
120
Relative activity of DNA synthesis(%)
100
(+/+)
80 60 40
(+/-)
20 0
0
1
2
3
4
SeqA (µg)
5
6
7
80 60 40 20 0
0
1
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3
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7
SeqA (µg)
120
Relative activity of DNA synthesis (%)
(B)
100
Relative activity of DNA synthesis(%)
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D. Douraid, L. Ahmed / Plasmid 65 (2011) 15–19
Fig. 3. Effect of various concentrations of SeqA on DNA synthesis from the hemimethylated oripBR322 plasmid DNA in the presence of 4 ll of antiSeqA antibody. Solid triangles (N), hemimethylated oripBR322 DNA; Solid squares (j), hemimethylated oripBR322 DNA supplemented with antiSeqA;
(+/+)
100 80 60 40
(+/-)
20 0
0
1
2
3
4
5
6
7
SeqA (µg) Fig. 2. Effect of SeqA on oriC and oripBR322 plasmid replication in vitro: (A) Inhibition of oriC plasmid DNA replication by various concentrations of SeqA. Standard conditions (5 lg of protein DnaA and without KCl) for in vitro replication of the oriC plasmid pOC42 were used. In the absence of SeqA, the amount of DNA synthesized was 176 pmol with hemimethylated template DNA and 272 pmol with fully methylated template DNA. (s), fully oriC methylated DNA (+/+); (D), hemimethylated oriC DNA (+/ ). (B) Inhibition of oripBR322 plasmid DNA replication by various concentrations of SeqA. The DnaA protein was substituted by 100 mM KCl. In the absence of SeqA, the amount of DNA synthesized was 292 pmoles with hemimethylated template DNA and 276 pmoles with fully methylated template DNA. (d) fully oripBR322 methylated DNA (+/ +); (N) hemimethylated oripBR322 DNA (+/ ).
examined the effect of the addition of the SeqA-antibody to the replication mixture. The addition of the anti-SeqA antibody during the in vitro replication, at the oripBR322 region, blocks specifically the effect of the SeqA protein (Fig. 3). As a result, in this condition, the amount of radioactivity labeled desoxynucleotides incorporated into newly synthesized DNA, was measured. This amount is similar to the amount of radioactivity obtained with the replication starting from an oripBR322 methylated DNA state (Fig. 3). These findings confirm that the inhibition of the initiation of the replication at oripBR322 sequence requires a direct interaction between the SeqA protein and the hemimethylated RNA II promoter region of pBR322.
4. Discussion In this study, we show that hemimethylation-mediated inhibition of pBR322 replication relies on the same mechanism previously described for oriC, i.e., response to adenine methylation (Russell and Zinder, 1987) and the sequestration of the hemimethylated origin region away from initiation by the cell membrane (Malki et al., 1991).
On the basis of earlier studies (Ogden et al., 1988; Landoulsi et al., 1990), Lu et al. (1994) proposed, that hemimethylated oriC binds specifically to a high density membrane fraction in vitro, that SeqA acts negatively on the initiation of replication to sequestrate oriC into the membrane where it is transiently refractory to re-initiation. In vivo, two different effects of SeqA on DNA replication have been distinguished. First, SeqA delays replication initiation without perturbing the precision with which initiation is coupled to cell physiology (Boye et al., 1996). Second, SeqA is required for sequestration of oriC from re-methylation immediately following replication initiation and, in a presumably related effect, for blocking replication of hemimethylated oriC plasmids (Lu et al., 1994; Slater et al., 1995; Taghbalout et al., 2000) and the oriP plasmid (Brendler et al., 1995). As plasmids driven by oripBR322 are also subject to sequestration (Landoulsi et al., 1990; Malki et al., 1991), it is probable that the SeqA protein regulates pBR322 replication of newly synthesized DNA by detecting its hemimethylated state. In vitro replication of pBR322 involves probably sequestration of the RNA II primer promoter away from RNA polymerase by SeqA with a consequential reduction in RNA II primer transcription and initiation frequency at the downstream start site for DNA synthesis. Our results indicate that the SeqA recognition of the RNA II promoter region of pBR322 involves direct DNA binding of the SeqA protein to the hemimethylated origin sequences formed as a result of replication. Thus, SeqA would block initiation directly by preventing binding of essential factors or by interfering with some key event (Waldminghaus and Skarstad, 2009), such as strand opening. This would prevent re-initiation until the unmethylated strands are methylated, thus releasing the SeqA protein. Acknowledgments We are most grateful to Dr. M. Kohiyama for providing bacterial strains, without which this work would not be possible.
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