Department of Molecular Biology, University of Gdask1 and Laboratory of Molecular Biology (affiliated with the University of Gda
sk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences2, K
adki 24, 80-822 Gda
sk, Poland
Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas (CSIC), Velázquez 144, 28006 Madrid, Spain3
Center for Molecular Genetics and Department of Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA4
Marine Biology Center, Polish Academy of Sciences, w. Wojciecha 5, 81-347 Gdynia, Poland5
Author for correspondence: Grzegorz Wgrzyn. Tel: +48 58 346 3014. Fax: +48 58 301 0072. e-mail: wegrzyn{at}biotech.univ.gda.pl
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ABSTRACT |
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Keywords: bacteriophage development, Escherichia coli DnaA protein, rolling-circle DNA replication, theta DNA replication, transcriptional activation of origin
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INTRODUCTION |
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The genome of bacteriophage consists of 48502 bp of double-stranded DNA (see Fig. 1
). In the
virion, this genetic material is packaged in a linear form in the head of the phage capsid. After adsorption to the surface of its host, Escherichia coli, and penetration of the phage genome into the bacterial cell, the linear DNA is immediately converted into the circular form due to single-stranded 5' extensions of 12 bases at both ends, which are complementary to each other. The ends are ligated by the host DNA ligase and, following the action of E. coli DNA gyrase, the phage genome becomes a negatively supercoiled structure (for recent reviews on
DNA replication and phage development see Taylor & W
grzyn, 1995
, 1998
).
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After five to six rounds of bidirectional replication, about 50 copies of the circular
genome appear in the infected host cell. Then, at about 15 min post-infection, a few of these molecules start to replicate according to the
mode (Taylor & W
grzyn, 1995
, 1998
). This leads to the production of long concatemers of
DNA, up to about ten genome equivalents in length, which are cut at specific sites (called cos sites) and serve as the substrates for the phage packaging system. The mechanism of the switch from
to
replication has not been completely elucidated, but according to the predominant hypothesis, originally proposed by Dodson et al. (1986
),
may be preceded by one round of unidirectional
replication initiated at ori
, followed by displacement of the 5' end of the newly synthesized leading strand by its growing 3' end.
Although the mechanism of the switch from to
replication has been proposed (see above), it remains unknown how this process is regulated and what triggers the change in the replication mode of
DNA. We have found previously that the host dnaA gene function has a role in phage
replication (G. W
grzyn et al., 1995b
). The findings that (i) DnaA stimulates transcriptional activation of ori
(G. W
grzyn et al., 1995a
; Szalewska-Pa
asz et al., 1998a
); (ii) transcription is necessary for bidirectional
replication of
DNA in vitro (Learn et al., 1993
); (iii) unidirectional
replication is considered as a prerequisite for
replication (Dodson et al., 1986
); and (iv)
replication intermediates of the phage bearing mutations in the P gene appear early after infection of the dnaA host (G. W
grzyn et al., 1995b
; Konopa et al., 2000
), suggested that DnaA may be a crucial factor in triggering the switch from
to
replication of phage
DNA in infected E. coli cells. Therefore, the aim of this work was to investigate the mechanism of regulation of this switch in wild-type
phage and the role of the DnaA protein in this process.
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METHODS |
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One-step growth experiments.
Lytic development of bacteriophage was investigated by one-step growth experiments as described previously (G. W
grzyn et al., 1995b
). Briefly, bacteria growing exponentially in LB medium at 30 °C were infected by phage
cIb2 in the presence of 3 mM NaN3 (to prevent unsynchronized phage development) at the indicated m.o.i., and adsorption was carried out for 10 min at 43 °C. Following centrifugation, the bacterial pellet was resuspended in the same medium (with NaN3) containing anti-
serum and incubated for 5 min at 43 °C to neutralize unadsorbed phages. The suspension was then diluted 1000-fold with prewarmed (to 43 °C) medium (devoid of NaN3) and aerated in a water-bath shaker. The number of infective centres was estimated by plating samples taken during the first 10 min after dilution (time 010 min). In fact, the estimated number of infective centres is the sum of the number of infected bacteria and the number of free, unadsorbed phages. However, as most of the unadsorbed phage particles were neutralized by anti-
serum, the second value was ignored during the calculation of the burst size. The number of intracellular progeny phage was estimated by plating chloroform-treated samples of the infected culture withdrawn at different times, using strain MG1655 as a host. The burst size was calculated as the ratio of the number of progeny phages to the number of infective centres.
Density-shift experiments.
These were performed according to G. Wgrzyn et al. (1995b
). Briefly, in the first type of experiment, bacteria were grown in a light minimal medium (W
grzyn, G. et al., 1995b
) overnight at 30 °C, and after dilution (1:50, v/v) with fresh medium the growth was continued to an OD500 of 0·2. The bacteria were pelleted, washed with TM buffer (10 mM Tris/HCl, pH 7·2, 10 mM MgSO4) and suspended in 0·1 vol. of this buffer. After 60 min incubation at 43 °C, [3H]thymidine-labelled phage (8·3x10-5 c.p.m. p.f.u.-1) was added to a m.o.i. of 10 and incubation was continued for 15 min. The suspension was sedimented, resuspended in the original volume of prewarmed (43 °C) heavy minimal medium (containing 15NH4Cl and [13C]glucose instead of NH4Cl and glucose, respectively), and further incubation was performed at 43 °C. Samples of the infected culture were withdrawn at the indicated times, and total DNA was isolated and ultracentrifuged in a CsCl density gradient as described previously (G. W
grzyn et al., 1995b
). Fractions were collected from the bottom of the tube and the radioactivity of each fraction was measured in a scintillation counter. In the second type of experiment, the procedure was the same as described above but bacteria growing in the light medium were infected with unlabelled phage (m.o.i. of 10) and further incubation was continued in the heavy medium containing 0·1 mCi [3H]thymidine ml-1.
Measurement of total DNA synthesis.
Synthesis of DNA in cells was investigated by measurement of incorporation of [3H]thymidine into trichloroacetic acid (TCA)-precipitable material according to the method described by Wgrzyn et al. (1991
). Briefly, the bacterial cultures were labelled with 0·1 mCi [3H]thymidine ml-1. Samples were withdrawn at the indicated times and transferred onto paper filters. The filters were placed immediately in 10% ice-cold TCA for 5 min, and transferred to 5% TCA for 5 min. The samples were then washed twice with 96% ethanol and dried at room temperature. The radioactivity of the samples was measured in a scintillation counter.
Two-dimensional agarose gel electrophoresis.
Analysis of replication intermediates by two-dimensional agarose gel electrophoresis was performed according to Viguera et al. (1996 ), with modifications described by
rutkowska et al. (1999
).
Electron microscopy.
Isolation of plasmid DNA was performed according to Viguera et al. (1996 ) and electron microscopy analysis of replicating plasmid DNA molecules was performed as described by Burkardt & Lurz (1984
) and
rutkowska et al. (1998
).
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RESULTS |
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Phage DNA replication in E. coli dnaA+ and dnaA hosts
To investigate phage DNA replication in infected cells we employed density-shift experiments. The cells growing in a light medium were mixed with phage lysate previously labelled with [3H]thymidine, and after adsorption the cultivation was continued in a heavy medium. We found that the adsorption efficiency of the phage on both dnaA+ and dnaA46(ts) hosts was very similar in these conditions, with a difference below 5%. The fate of parental phage DNA was monitored by isolation of total DNA and ultracentrifugation in a caesium chloride density gradient. The recovery of labelled phage DNA was similar in both strains: 31% in dnaA+ and 25% in dnaA46(ts), on average. In these experiments we used the
red3 mutant to impair recombination between phage DNA molecules. When wild-type (MG1655; dnaA+) cells were infected, the phage DNA (originally all of it located at the full-light position, data not shown) was found in the full-light (non-replicated molecules) and heavylight (molecules after at least one replication round) positions 5 and 15 min after infection (Fig. 3a
). All radioactivity moved to the heavylight fractions at 30 min, where it remained until the end of the experiment (60 min). Since we monitored the fate of parental phage DNA (labelled with [3H]thymidine), several rounds of replication proceeding by the
mode would result in the shift of radioactivity only to the heavylight position. At later times after infection
replication intermediates should appear and if parental DNA molecules were to enter this mode of replication, a further shift towards full-heavy position should be observed (total length of
intermediates is up to about ten
genomes; thus, if such a molecule contained one strand of light 3H labelled DNA of the length of one unit of the genome and the rest of the duplex DNA structure were composed of heavy nucleotides, it should be located close to the full-heavy position). However, since on average only a few of about 50
DNA circles that appear due to
replication enter rolling-circle replication (see Taylor & W
grzyn, 1995
), no significant shift towards the full-heavy position was observed (Fig. 3a
). Different results were obtained when the dnaA46(ts) bacteria were infected at 43 °C. The shift towards the full-heavy position was already observed 15 min post-infection and it was much more significant at later times (Fig. 3a
). This shift strongly suggests that a larger fraction of parental phage DNA molecules enter
replication at this temperature.
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Phage DNA synthesis in E. coli dnaA+ and dnaA46(ts) hosts at 43 °C
The results presented in the preceding paragraphs suggested that contrary to the -infected wild-type host, the early round(s) of replication of phage
DNA in the dnaA mutants may proceed predominantly by the
mode. Therefore, on the basis of the previously proposed model concerning the mechanism of the switch from early to late
DNA replication (Dodson et al., 1986
), one may speculate that early after infection of the host devoid of DnaA,
DNA replicates according to the unidirectional
mode, which is followed by
replication. To test this hypothesis, we repeated the density-shift experiments except that a non-labelled phage was used. Bacteria growing in the light medium were infected with light phage and further incubation was continued in the heavy medium containing [3H]thymidine. Several rounds of
replication of the infecting phage genome should lead to the appearance of radioactive DNA at the full-heavy position relatively shortly after infection. Up to 30 min after the shift from light to heavy medium, one replication round of the host chromosome is possible at most, thus any radioactivity found in the full-heavy fraction must come from newly synthesized
DNA. We determined that under the conditions used in these experiments, incorporation of [3H]thymidine into TCA-precipitable material was effective during the first 15 min, and later the efficiency of incorporation dropped significantly (Fig. 4
), most probably due to exhaustion of radioactive thymidine. As samples for DNA isolation and density-gradient centrifugation were withdrawn 15 and 30 min post-infection, radioactivity in the full-heavy fractions observed after the first time interval represented
DNA molecules synthesized according to the
mode, and that observed after the second time interval indicated the fate of
DNA already synthesized during the first 15 min post-infection. We found considerable radioactivity in the full-heavy fractions in samples of the culture of the
-infected dnaA+ host withdrawn 15 min post-infection (Table 1
), indicating that several rounds of
replication had occurred. However, low activity was observed in analogous fractions in samples of the culture of the dnaA46(ts) host infected with
at 43 °C (Table 1
). Therefore, it may be suggested that at most one round of
replication was possible in the case of a predominant number of infecting phage DNA molecules. Effective
DNA replication in the dnaA46(ts) mutant during the first 15 min was confirmed by monitoring the distribution of radioactivity in samples withdrawn 30 min post-infection. Significant radioactivity in the full-heavy fractions was found, perhaps representing
replication intermediates (Table 1
). Incorporation of radioactivity into DNA in the heavylight fractions (mostly E. coli chromosomal DNA) was only slightly lower in the dnaA46(ts) host than in the wild-type strain (15 min post-infection the radioactivity in heavylight fractions in the sample from the dnaA46(ts) bacteria was 71% of that found in the sample from the wild-type bacteria, and 30 min postinfection, the radioactivity in heavylight fractions in the sample from the dnaA46(ts) bacteria was 86% of that found in the sample from the wild-type bacteria). Moreover, we observed no shift of radioactivity to the full-heavy position in analogous samples from non-infected cultures (data not shown).
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Directionality of replication of
DNA analysed by two-dimensional agarose gel electrophoresis
Plasmids derived from bacteriophage (referred to as
plasmids) contain all the genes and regulatory sequences necessary for autonomous replication in E. coli. These plasmids replicate in the host cells by the
mode (for reviews see Taylor & W
grzyn, 1995
, 1998
). Therefore, we used the
plasmids in studies on the directionality of
replication of
DNA in dnaA+ and dnaA46(ts) strains. Wild-type
plasmids cannot be maintained in certain dnaA(ts) mutants (including dnaA46) even at 30 °C, but derivatives bearing a mutation of the
type in the
P gene can (Kur et al., 1987
; W
grzyn et al., 1996
). Thus, in experiments with a dnaA46(ts) host we used the
mutant plasmid. Since it was demonstrated previously that even
plasmids can perform only one replication round in the dnaA46(ts) host after a shift from 30 °C to 43 °C (Kur et al., 1987
), we isolated plasmid DNA for two-dimensional agarose gel electrophoresis analysis 15 min after the temperature shift.
We used E. coli dnaA+ and dnaA46(ts) hosts bearing plasmids pKB2 and pKB2, respectively. Following isolation, these plasmids were digested with different restriction enzymes (shown in Fig. 1b
) and analysed by two-dimensional agarose gel electrophoresis. Theoretical patterns of
plasmid replication intermediates in the case of bidirectional and unidirectional (leftward and rightward) replication in samples digested with HindIII and BamHI, predicted by a computer method assuming that replication forks initiate synchronously and travel at the same rate (Viguera et al., 1998
), are presented in Fig. 5(a)
. The results obtained and their interpretation are presented in Fig. 5(b)
. In the dnaA+ host, replication of wild-type
plasmid (pKB2) proceeds both bidirectionally and unidirectionally. Bidirectional replication is represented by the characteristic shape of the bubble arc and the intensive descending arm of the simple-Y arc. The dots at the top and at the ascending arm of the simple-Y arc suggest rightward and leftward unidirectional replication, respectively. No significant differences in the pattern of replication intermediates were observed between DNA samples isolated from dnaA+/pKB2 bacteria growing at different temperatures: 30 °C, 37 °C or 43 °C (data not shown). Similar to the wild-type plasmid in the dnaA+ host, replication of pKB2
in dnaA46 cells at 30 °C proceeds both bidirectionally and unidirectionally. In the case of unidirectional replication, the intermediates of both leftward and rightward replication were detected. Thus, the presence of the
mutation in the plasmid and dnaA46(ts) mutation in the host genome has no significant influence on the directionality of plasmid replication at 30 °C. However, analysis of pKB2
replication intermediates isolated from the dnaA46(ts) mutant growing at 43 °C revealed that bidirectional replication was less frequent relative to that observed at 30 °C as the descending arm of the simple-Y arc is considerably less intensive.
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Electron microscopic analysis of the directionality of replication of
DNA
Two-dimensional agarose gel electrophoresis demonstrated that an impaired DnaA function may result in less frequent bidirectional replication of plasmid DNA. To obtain quantitative data, we analysed plasmid molecules (isolated as for two-dimensional agarose gel electrophoresis) using electron microscopic techniques. Plasmid DNA molecules isolated from dnaA+ or dnaA46(ts) bacteria were cut with HindIII and BamHI (see Fig. 1b
) and analysed by electron microscopy. Fragments of molecules containing bubbles as well as Y-shaped DNA fragments were identified and the lengths of appropriate arms were measured. For determination of the directionality of replication, the known position of ori
was assumed to be the only possible replication start point (Fig. 6
). Thus, we could determine the fraction of bidirectionally and unidirectionally replicating plasmids. We found that in the dnaA+ host about 40% of
plasmid molecules replicate bidirectionally irrespective of temperature (Table 2
). A similar distribution of plasmid replication intermediates was found in the dnaA46(ts) mutant growing at 30 °C, confirming the conclusion based on two-dimensional agarose gel analysis that combination of
and dnaA46(ts) mutations has little effect on directionality of
plasmid replication at this temperature. However, among all replication intermediates found in dnaA46(ts) bacteria at 43 °C, only about 10% were derived from bidirectionally replicating plasmids (Table 2
). These results are in agreement with our conclusion, based on data from two-dimensional agarose gel electrophoresis, that bidirectional DNA replication from ori
is impaired in the absence of functional DnaA.
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DISCUSSION |
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By analysis of replication initiated at ori, performed using two dimensional agarose gel electrophoresis and electron microscopy, we demonstrated that while in the wild-type host and in the dnaA46(ts) mutant at 30 °C bidirectional replication is frequent, in the dnaA46(ts) host at 43 °C it is predominantly unidirectional. Since replacement of the pR promoter with ptet (whose activity is not dependent on DnaA) resulted in abolition of the effect of the dnaA mutation on the replication directionality, we conclude that DnaA acts in the control of directionality of replication from ori
by regulation of transcription initiated at pR.
Model for regulation of the switch from to
DNA replication
Transcription initiated at the pR promoter and proceeding near or through ori is called transcriptional activation of ori
. This process has been known for a long time (Dove et al., 1969
; Nijkamp et al., 1971
) to be necessary for
DNA replication in vivo, but its exact role was unclear until recently. The results presented in this paper, together with previously reported observations (Learn et al., 1993
; G. W
grzyn et al., 1995b
, 1996
), indicate that transcriptional activation of ori
is necessary for initiation of bidirectional replication from this region. This led us to propose a mechanism of regulation of the switch from
to
replication of bacteriophage
DNA. Since DnaA positively regulates transcription from the pR promoter (Szalewska-Pa
asz et al., 1998a
), its activity is necessary for frequent initiation of bidirectional replication from ori
in E. coli. Early after infection, in spite of the presence of many DnaA boxes in the E. coli chromosome, there is a sufficient concentration of free DnaA molecules in the cell to stimulate bidirectional
replication, as maximal activation of pR by DnaA occurs at relatively low concentrations of this protein (Szalewska-Pa
asz et al., 1998a
). However, after a few rounds of bidirectional
replication, many copies of the
genome appear (about 50 copies after five to six replication rounds). Since it was previously demonstrated by Szalewska-Pa
asz et al. (1998c
) that there are many DnaA binding sites in
DNA, the DnaA protein may be titrated out. This should lead to an inefficient transcriptional activation of ori
resulting in unidirectional
replication followed by the
replication mode. In dnaA mutants, weak transcription from the pR promoter allows predominantly unidirectional
replication and a switch to
replication shortly after infection. This model is presented schematically in Fig. 7
. If this model is correct, the DnaA box sequences responsible for titrating out DnaA molecules after several rounds of bidirectional
replication of the
genome in the wild-type host should be of weak affinity to this protein. This would allow efficient transcriptional activation of ori
and bidirectional
replication at the beginning of the infection cycle. In accordance with this prediction, it was reported that most DnaA boxes present in the
genome are weak ones (Szalewska-Pa
asz et al., 1998c
).
Better & Freifelder (1983 ) reported that
replication intermediates can be also occasionally found at early times after infection of wild-type hosts by
phage. They found that
replication is predominant, but not exclusive, at early stages of infection and
replication is predominant, but not exclusive, at late stages of infection. This is also compatible with the results presented in this report and with our model of the switch from
to
replication, as we have demonstrated that in the presence of DnaA function the replication of
plasmids proceeds bidirectionally and unidirectionally, and in the absence of DnaA function the replication is predominantly, but not exclusively, unidirectional. In fact, after inactivation of DnaA we observed (using electron microscopy) a decrease in the fraction of bidirectionally replicating
DNA from 40% to 10%, whereas the dnaA46(ts) mutation at 43 °C was responsible for a huge increase in
-type replication intermediates as revealed by density-shift experiments. However, one should note that the electron microscopy studies were performed with plasmids occurring in many copies per cell, whereas in the density shift experiments only a few phages infected one cell. Moreover, whole population of
plasmid molecules was investigated by electron microscopy, whereas the fate of only parental phage
DNA molecules was monitored in density-shift experiments.
A physiological role for the switch from to
DNA replication
It is also interesting to consider a physiological role for a DnaA-regulated switch from to
replication of phage
DNA. Since efficient growth of
phage was observed in the dnaA46(ts) mutant at 43 °C, i.e. under conditions in which
replication starts early after infection, it raises the question as why should
replication occur at early stages of infection in a wild-type host? The physiological role of
replication should be the production of many copies of
DNA to be used as templates for high level of expression of phage genes employed during lytic development. If this is true, the amount of
DNA produced by
replication is high enough for expression of phage proteins in amounts sufficient to support normal lytic development. However, it is worth noting that studies on
development, which demonstrated efficient production of phage progeny in dnaA mutants (G. W
grzyn et al., 1995b
; Sutton & Kaguni, 1997
; Szalewska-Pa
asz et al., 1998b
; Glinkowska et al., 1999
; this work), were carried out under standard laboratory conditions (i.e. in LB medium with good aeration, etc.) that support high E. coli growth rates. It was demonstrated recently that under these conditions, in a wild-type host, phage
produces at least some proteins in excess of those needed for its effective propagation: thus efficient growth of the phage may be achieved even under less favourable conditions (Gabig et al., 1998
). This phage developmental strategy may be responsible for efficient
growth in dnaA mutants, in which expression of phage genes is expected to be decreased relative to the wild-type host. If this is true, one might expect an inhibition of
lytic development in a dnaA mutant growing in poor media. Indeed, it has been reported that phage
lytic growth is less efficient in wild-type cells cultivated in minimal media, and it may be completely inhibited in slowly growing dnaA46(ts) mutants at 43 °C (W
grzyn et al., 2000
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Alfano, C. & McMacken, R.(1989b). Heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage DNA replication. J Biol Chem 264, 10709-10718.
Better, M. & Freifelder, D.(1983). Studies on the replication of Escherichia coli phage DNA. Virology 126, 168-182.[Medline]
Burkardt, H. & Lurz, R.(1984). Electron microscopy. In Advanced Molecular Genetics , pp. 281-313. Edited by A. Puhler & K. N. Timmis. Berlin and Heidelberg:Springer.
Dodson, M., Echols, H., Wickner, S., Alfano, C., Mensa-Wilmot, K., Gomes, B., LeBowitz, J., Roberts, J. D. & McMacken, R.(1986). Specialized nucleoprotein structures at the origin of replication of bacteriophage : localized unwinding of duplex DNA by a six protein reaction. Proc Natl Acad Sci USA 83, 7638-7642.[Abstract]
Dove, W. F., Hargrove, E., Ohashi, M., Haugli, F. & Guha, A. (1969). Replicator activation in lambda. Jpn J Genet 44 (Suppl. 1), 1122.
Gabig, M., Obuchowski, M., Wgrzyn, A., Szalewska-Pa
asz, A., Thomas, M. S. & W
grzyn, G.(1998). Excess production of phage
delayed early proteins under conditions supporting high Escherichia coli growth rates. Microbiology 144, 2217-2224.[Abstract]
Glinkowska, M., Wgrzyn, A. & W
grzyn, G.(1999). Replication of bacteriophage
in the Escherichia coli dnaA
rac hosts. Genetics 151, 1633-1635.
Gottesman, M.(1999). Bacteriophage : the untold story. J Mol Biol 293, 177-180.[Medline]
Herman-Antosiewicz, A., rutkowska, S., Taylor, K. & W
grzyn, G.(1998a). Replication and maintenance of
plasmids devoid of the Cro repressor autoregulatory loop in Escherichia coli. Plasmid 40, 113-125.[Medline]
Herman-Antosiewicz, A., Wgrzyn, A., Taylor, K. & W
grzyn, G.(1998b). DnaA-mediated regulation of phage
-derived replicons in the absence of pR and Cro function. Virology 249, 98-107.[Medline]
Ingmer, H. & Atlung, T.(1992). Expression and regulation of a dnaA homologue isolated from Pseudomonas putida. Mol Gen Genet 232, 431-439.[Medline]
Jensen, K. F.(1993). The Escherichia coli wild types W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels. J Bacteriol 175, 3401-3407.[Abstract]
Konopa, G., Baraska, S., W
grzyn, A. & W
grzyn, G.(2000). Bacteriophage and host mutants causing the rolling-circle
DNA replication early after infection. FEBS Lett 472, 217-220.[Medline]
Kornberg, A. & Baker, T. A. (1992). DNA Replication. New York: W. H. Freeman.
Kur, J., Górska, I. & Taylor, K.(1987). Escherichia coli dnaA initiation function is required for replication of plasmids derived from coliphage lambda. J Mol Biol 198, 203-210.[Medline]
Learn, B., Karzai, A. W. & McMacken, R.(1993). Transcription stimulates the establishment of bidirectional DNA replication in vitro. Cold Spring Harbor Symp Quant Biol 58, 389-402.[Medline]
Mensa-Wilmot, K., Seaby, R., Alfano, C., Wold, M. S., Gomes, B. & McMacken, R.(1989). Reconstitution of a nine-protein system that initiates bacteriophage DNA replication. J Biol Chem 264, 2853-2861.
Nijkamp, H. J. J., Szybalski, W., Ohashi, M. & Dove, W. F.(1971). Gene expression by constitutive mutants of coliphage lambda. Mol Gen Genet 114, 80-88.
Schnos, M. & Inman, R. B.(1970). Position of branch point in replicating DNA. J Mol Biol 51, 61-73.[Medline]
Singer, M., Baker, T. A., Schnitzler, G. & 7 other authors (1989). A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev 53, 124.[Medline]
rutkowska, S., Konopa, G. & W
grzyn, G.(1998). A method for isolation of plasmid DNA replication intermediates from unsynchronized bacterial cultures for electron microscopy analysis. Acta Biochim Pol 45, 233-240.[Medline]
rutkowska, S., Caspi, R., Gabig, M. & W
grzyn, G.(1999). Detection of DNA replication intermediates after two-dimensional agarose gel electrophoresis using a fluorescein-labeled probe. Anal Biochem 269, 221-222.[Medline]
Stillman, B.(1994). Initiation of chromosomal DNA replication in eukaryotes: lessons from lambda. J Biol Chem 269, 7047-7050.
Subak-Sharpe, J. H. & Dargan, D. J.(1998). HSV molecular biology: general aspects of Herpes Simplex Virus molecular biology. Virus Genes 16, 239-251.[Medline]
Sutton, M. D. & Kaguni, J. M.(1997). Novel alleles of the Escherichia coli dnaA gene. J Mol Biol 271, 693-703.[Medline]
Szalewska-Paasz, A., W
grzyn, A., B
aszczak, A., Taylor, K. & W
grzyn, G.(1998a). DnaA-stimulated transcriptional activation of ori
: Escherichia coli RNA polymerase ß subunit as a transcriptional activator contact site. Proc Natl Acad Sci USA 95, 4241-4246.
Szalewska-Paasz, A., Lemieszek, E., Pankiewicz, A., W
grzyn, A., Helinski, D. R. & W
grzyn, G.(1998b). Escherichia coli dnaA gene function and bacteriophage
replication. FEMS Microbiol Lett 167, 27-32.[Medline]
Szalewska-Paasz, A., Weigel, C., Speck, C. & 7 other authors (1998c). Interaction of the Escherichia coli DnaA protein with bacteriophage
DNA. Mol Gen Genet 259, 679688.[Medline]
Taylor, K. & Wgrzyn, G.(1995). Replication of coliphage lambda DNA. FEMS Microbiol Rev 17, 109-119.[Medline]
Taylor, K. & Wgrzyn, G.(1998). Regulation of bacteriophage
replication. In Molecular Microbiology , pp. 81-97. Edited by S. J. W. Busby, C. M. Thomas & N. L. Brown. Berlin and Heidelberg:Springer.
Thomas, R.(1993). Bacteriophage : transactivation, positive control and other odd findings. BioEssays 15, 285-289.[Medline]
Viguera, E., Hernandez, P., Krimer, D. B., Boistov, A. S., Lurz, R., Alonso, J. C. & Schvartzman, J. B.(1996). The ColE1 unidirectional origin acts as a polar replication fork pausing site. J Biol Chem 271, 22414-22421.
Viguera, E., Rodríguez, A., Krimer, D. B., Hernández, P., Trelles, O. & Schvartzman, J. B.(1998). A computer model for the analysis of DNA replication intermediates by two-dimensional (2D) agarose gel electrophoresis. Gene 217, 41-49.[Medline]
Wgrzyn, A., W
grzyn, G. & Taylor, K.(1995a). Protection of coliphage
O initiator protein from proteolysis in the assembly of the replication complex in vivo. Virology 207, 179-184.[Medline]
Wgrzyn, A., W
grzyn, G. & Taylor, K.(1995b). Plasmid and host functions required for
plasmid replication carried out by the inherited replication complex. Mol Gen Genet 247, 501-508.[Medline]
Wgrzyn, A., Czy
, A., Gabig, M. & W
grzyn, G.(2000). ClpP/ClpX-mediated degradation of the bacteriophage
O protein and regulation of
phage and
plasmid replication. Arch Microbiol 174, 89-96.[Medline]
Wgrzyn, G., Kwásnik, E. & Taylor, K.(1991). Replication of
plasmid in amino acid-starved strains of Escherichia coli. Acta Biochim Pol 38, 181-186.[Medline]
Wgrzyn, G., Szalewska-Pa
asz, A., W
grzyn, A., Obuchowski, M. & Taylor, K.(1995a). Transcriptional activation of the origin of coliphage
DNA replication is regulated by the host DnaA initiator function. Gene 154, 47-50.[Medline]
Wgrzyn, G., W
grzyn, A., Konieczny, I., Bielawski, K., Konopa, G., Obuchowski, M., Helinski, D. R. & Taylor, K.(1995b). Involvement of the host initiator function dnaA in the replication of coliphage
. Genetics 139, 1469-1481.
Wgrzyn, G., W
grzyn, A., Pankiewicz, A. & Taylor, K.(1996). Allele specificity of the Escherichia coli dnaA gene function in the replication of plasmids derived from phage
. Mol Gen Genet 252, 580-586.[Medline]
ylicz, M., Ang, D., Liberek, K. & Georgopoulos, C.(1989). Initiation of
DNA replication with purified host- and bacteriophage-encoded proteins: the role of the DnaK, DnaJ and GrpE heat shock proteins. EMBO J 8, 1601-1608.[Abstract]
Received 1 September 2000;
revised 13 November 2000;
accepted 27 November 2000.