Department of Life Sciences and Chemistry, 18-1, Roskilde University, PO Box 260, DK-4000 Roskilde, Denmark
Correspondence
Ole Skovgaard
olesk{at}ruc.dk
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ABSTRACT |
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INTRODUCTION |
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Initiation of E. coli chromosome replication is coupled to cell growth in such a way that it occurs when a certain cell mass per origin, the initiation mass, has accumulated (Donachie, 1968). The initiation mass is fairly constant over a wide range of growth rates (reviewed by Bipatnath et al., 1998
) and is mainly set by accumulation of the DnaA protein in wild-type cells (Løbner-Olesen et al., 1989
). In different dnaA mutants the initiation mass is increased, indicating that the reduced activity of the DnaA protein is compensated for by a decreased concentration of origins in the cell (Boye et al., 1996
).
In the initiation process, the DnaA protein bound to either ATP or ADP recognizes and binds to its five DnaA-boxes in oriC (Fuller et al., 1984). Subsequently, ATP-bound DnaA recognizes an additional set of 9-mer sequences (I-boxes; McGarry et al., 2004
). This triggers duplex opening in the AT-rich region, and DnaA-ATP stabilizes this open complex by binding 6-mer motifs in the single-stranded AT-rich region (Speck et al., 1999
). In the final stages of initiation, DnaA interacts with the DnaB helicase and recruits it to the open complex (Marszalek & Kaguni, 1994
; Seitz et al., 2000
) to form the pre-priming complex that facilitates further strand separation and allows for entry of the replication machinery.
Fast-growing E. coli cells contain multiple origins of replication, which are synchronously initiated, once and only once per cell cycle. Several mechanisms contribute to this stringent control of chromosome replication. Immediate reinitiation is prevented by sequestration of hemimethylated origins (Campbell & Kleckner, 1990). During sequestration at least three mechanisms operate to lower the activity of the DnaA initiator protein sufficiently for initiation only to occur one mass doubling later. First, the dnaA promoter is also sequestered (Lu et al., 1994
). This prevents de novo DnaA synthesis and accumulation during the sequestration period (Campbell & Kleckner, 1990
). Second, new DnaA-binding sites outside the origin are generated by replication. These sites serve to lower the amount of free DnaA protein available for initiation. The most prominent of these DnaA binding sites, datA, is located about 0·7 Mb away from oriC. The datA region consists of two active DnaA-boxes that titrate large amounts of DnaA protein (Kitagawa et al., 1998
; Ogawa et al., 2002
) presumably in either ATP- or ADP-bound form. Third, DnaA-ATP is converted to DnaA-ADP by a process called RIDA involving the sliding clamp of active replication forks as well as the Hda protein (Su'etsugu et al., 2004
).
E. coli cells with reduced DnaA protein activity, due either to certain mutations in domain III or IV of the protein, or to the introduction of additional datA sites, initiate replication at an increased cell mass per origin. Initiations are often asynchronous, indicating that not all origins are initiated each cell cycle. In such cells the time required to replicate the chromosome is reduced and cells contain only few active replication forks (Morigen et al., 2003).
The dnaX gene encodes both the and the
subunits of DNA polymerase III holoenzyme by a frame-shifting mechanism and consequently these proteins are identical for the first 430 amino acids (domains IIII) (Blinkova & Walker, 1990
; Flower & McHenry, 1990
). The intensive research on the role of
and
in formation of the DnaX complex was recently reviewed by McHenry (2003)
. The DnaX complex has a dual role in organizing the asymmetric dimeric replicase and loading the
2 clamp onto DNA. Domain I of
/
has similarity to the AAA+ class ATPases, and both
and
possess ATPase activity. Domains IV and V are only present in the longer
and are necessary for interaction with DnaB helicase and the core of DNA polymerase III. Two
subunits organize the replisome by connecting two cores with DnaB helicase, imparting coordinated leading and lagging strand synthesis and rapid fork movement. The
subunit appears to organize the other subunits of the DnaX complex and connects this complex to the two
subunits. The entire DnaX complex has the stoichiometry
2
1
1
'1
1
1 in the mature in vivo DNA polymerase III. The dnaX2016 temperature-sensitive mutation changes glycine 118 to aspartate in both
and
(Blinkova et al., 1993
). This glycine is located between the Box IV' and the Walker B motif in the AAA+ domain I (Neuwald et al., 1999
), and the dnaX2016 mutation potentially affects the ATPase activity of
/
.
Extragenic mutations that partially suppress the temperature-sensitive phenotype of dnaX2016 (Sx phenotype) are easily obtained. Some of these mutations reside in the dnaA gene and consequently a direct interaction between the DnaA and the DnaX proteins was suggested (Walker et al., 1982; Ginés-Candelaria et al., 1995
). Because these mutants were isolated for being concomitantly cold-sensitive, they are termed dnaA(Cs,Sx) mutants.
In this work seven new dnaX(Ts)-suppressing mutations in the dnaA gene are characterized. The amino acids changed in these mutants are widely distributed over domains III and IV of the DnaA protein. The initiation of replication is compromised in all the dnaA(Sx) mutants, implying that the mutant DnaA proteins are partly deficient in sustaining initiation of chromosomal DNA replication from oriC. Furthermore, all the dnaA(Sx) mutants alleviated the strong SOS response induced in the dnaX2016 mutant at non-permissive temperature.
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METHODS |
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Strains for measuring SOS induction were derived from RUC1282 or RUC1291 and strains for all other growth experiments were derived from RUC1024 by P1 transductions with selection for resistance to tetracycline [dnaA(Sx) alleles] or chloramphenicol (dnaX2016 mutation) and screening with restriction site polymorphism or with RG-PCR (Gasparini et al., 1992).
Plasmids pACYC177, pMW119 (a pSC101-based vector), pMOR6 (pACYC177-datA) and pMOR8 (pMW119-datA) were all obtained from Morigen (Morigen et al., 2001).
Bacterial cultures were grown in AB minimal medium supplemented with 0·1 µg thiamine ml1, 0·2 % glucose, 1 % Casamino acids (Difco) and 0·05 % serine (ABTG-caa medium).
Chromosomal gene replacement.
A part of the dnaA gene containing the dnaA(Sx) mutation was PCR-amplified with primers dnaA3 (GGCGGATAACCCTGGCG) and dnaA4 (CCACCTAACGGACCGCTCAC). The DNA fragment was used to replace the temperature-sensitive dnaA46 allele on the chromosome of RUC1227 (KM22, dnaA46, tna2123 : : Tn10) with Red-mediated recombination (Murphy, 1998
) by selection for growth at 42 °C. The replaced DNA fragment was amplified by PCR in candidates, screened for restriction site polymorphism or with RG-PCR (Gasparini et al., 1992
) and finally sequenced to ensure perfect gene replacement.
DNA sequencing.
Sequences were obtained with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and an ABI Prism 310 Genetic Analyser (Applied Biosystems).
Autorepression.
Strains derived from RUC1024 were grown exponentially at 34 °C in ABGT-caa medium for five doubling times and triplicate samples were taken at OD450 0·5 for measuring specific -galactosidase activity (Miller, 1992
) from the dnaAp''lacZ fusion in the attB site.
SOS induction.
Strains derived from RUC1282 or RUC1291 were grown exponentially at 34 °C in ABGT-caa medium for five doubling times. At OD450 0·5, cultures were diluted 10-fold into prewarmed medium at 38 °C for continuous growth at 38 °C. Duplicate samples for specific -galactosidase activity from the sulA''lacZ fusion were taken every 30 min. Cultures were diluted further into prewarmed medium when OD450 reached 0·5.
Quantity and stability of DnaA proteins.
Strains derived from RUC1024 were grown exponentially at 34 °C in ABTG-caa medium. At OD450 0·5, a t=0 sample was taken and chloramphenicol was added to 200 µg ml1 to two aliquots followed by incubation at either 34 °C or 42 °C. Samples from the chloramphenicol-treated aliquots were taken at 30, 60 and 120 min. Proteins were separated by SDS-PAGE and immunoblotted with DnaA antibody obtained from K. Skarstad (Institute for Cancer Research, Oslo, Norway). Alkaline phosphatase activity of the secondary antibody was visualized with ECF substrate (Amersham Biosciences), scanned with a STORM840 PhosphorImager (Amersham Biosciences), and quantified with TotalLab software (Nonlinear Dynamics/Amersham Biosciences).
Flow cytometry.
This was performed as described by Løbner-Olesen et al. (1989) on a Bryte-HS flowcytometer (Bio-Rad) equipped with a 100 W mercury lamp. Strains derived from RUC1024 were grown exponentially at 34 °C in ABTG-caa medium. At OD450 0·5, samples were incubated with rifampicin (300 µg ml1) and cephalexin (36 µg ml1) to prevent new initiations of DNA replication and cell division.
Determination of the replication time, C, and the number of replication forks per cell, Fc.
Chromosomal DNA was prepared, and the origin to terminus ratio (O/T) was determined by marker frequency analysis exactly as described by Atlung & Hansen (1999). When a culture has a doubling time
and a chromosomal replication time C, the stoichiometry between origins, O, and termini, T, is given by the formula O/T=2C/
(Bremer & Churchward, 1977
), from which C is extracted as C=ln(O/T)·
·[ln(2)1]. The number of active replication forks, Fc, is given by the formula Fc=2(IcTc) (Bremer & Churchward, 1977
), where Ic is the mean number of origins per cell and Tc is the mean number of termini per cell. Ic was determined by flow cytometry and Tc was determined by Ic divided by O/T.
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RESULTS |
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Previously isolated dnaX2016(Ts) suppressors residing in the dnaA gene were all selected by sensitivity to growth at 20 °C (Walker et al., 1982; Ginés-Candelaria et al., 1995
). None of the new mutants isolated as described above were cold-sensitive, demonstrating that cold-sensitivity and Sx phenotype are independent.
The dnaA(Sx) mutations reduce the SOS induction in a dnaX2016 strain
The role of the and
proteins in organizing the DNA polymerase III holoenzyme and loading of the
-clamp suggests that cells carrying the dnaX2016 mutation might suffer replication fork collapse at non-permissive temperatures and consequently induce the SOS response (Seigneur et al., 1998
). The SOS response was measured as
-galactosidase activity from a sulA''lacZ fusion carried by a
phage (Lin & Little, 1988
).
During steady-state growth at 34 °C, the amount of -galactosidase expressed from this fusion in wild-type cells was 62 units. The
-galactosidase activity of wild-type cells did not change upon a shift to 38 °C (Fig. 2
, filled squares). In cells carrying the dnaX2016 mutation the amount of
-galactosidase was 102 units, an indication of slight SOS induction even at permissive temperature. After shifting the dnaX2016 strain to 38 °C the
-galactosidase activity increased immediately; over time a fivefold increase was observed (Fig. 2
, open squares). When the dnaX2016 mutation was combined with either of the dnaA(Sx) mutations, the activity of the sulA''lacZ fusion was reduced to levels between 80 and 92 units, indicating a partial relief of the signal for SOS induction at permissive temperature. No or little increase in
-galactosidase activity upon a shift to 38 °C was observed for any of the dnaX2016 dnaA(Sx) double mutants (Fig. 2
).
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The dnaA(Sx) mutants have reduced autorepression
The E. coli dnaA gene is autoregulated by a mechanism in which the DnaA protein binds to a region between promoters p1 and p2 of the dnaA gene, thereby repressing transcription from both promoters (Braun et al., 1985). The expression of a dnaA gene transcriptionally fused to lacZ is therefore a convenient way of measuring DnaA proteinDnaA-box interaction in vivo (Braun et al., 1985
). A dnaA''lacZ gene fusion integrated in the
attachment site attB of the chromosome (Berenstein et al., 2002
) was used. This was convenient because the gene dosage of attB changes little with changing growth rates and/or replication times (C-periods). Transcription from dnaAp was increased for all of the new dnaA(Sx) mutants. The degree of derepression was in the range 1·52·0 fold (Table 1
).
The observed derepression of dnaA gene transcription could be due either to instability of the DnaA(Sx) proteins resulting in lowered DnaA protein concentration, or to a poor interaction with DnaA-boxes. The concentration and the stability of each DnaA(Sx) protein was therefore determined (Fig. 3, Table 1
). Most of the dnaA(Sx) mutations led to little or no change in DnaA protein concentration in the cells relative to wild-type. There was, however, one exception. The dnaA893 mutation had less than 50 % of wild-type DnaA protein concentration. The reduced DnaA concentration resulting from the dnaA893 allele was due to a greatly decreased stability of this protein because this protein was already absent 30 min after de novo protein synthesis was stopped with chloramphenicol (Fig. 3
). Wild-type DnaA protein as well as the remaining DnaA(Sx) proteins were stable over a 120 min period (Fig. 3
).
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The dnaA(Sx) mutants have increased initiation mass
The reduced ability to repress dnaA gene transcription suggests that the mutant DnaA(Sx) proteins may also interact poorly with the DnaA-boxes located in oriC, leading to feeble initiation of chromosome replication.
A flow cytometric analysis of cells treated with rifampicin and cephalexin (for details see Methods) revealed that the number of origins per cell was 5·9 for both dnaX+ and dnaX2016(Sx) mutant cells at permissive temperature (Table 2). All dnaA(Sx) mutations reduced this number of origins per cell by 1530 % in combination with the dnaX+ allele and by 2040 % in combination with the dnaX2016 allele (Table 2
). The mean cell mass was also determined for each culture (Table 2
) and this value was used to calculate the mean cell mass per origin. The mean cell mass per origin is equal to the initiation mass of the cells multiplied by ln 2 (Bremer et al., 1979
) and is therefore a reliable measure of the relative initiation mass. All dnaA(Sx) mutants had an increased initiation mass (Table 2
). The increase in initiation mass was around 50 % for most mutants but smaller (823 %) in the dnaA893 mutant. This clearly demonstrates that all dnaA(Sx) mutations resulted in reduced initiation efficiency that led to delayed initiation in the cell cycle.
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The low origin content of the dnaA(Sx) cells also indicated that these cells contained fewer replication forks than their wild-type counterparts. The mean number of replication forks per cell in exponentially growing cells, Fc, is a function of the replication time, C, the time from termination of replication to cell division, D, and the doubling time, , of the culture (Bremer & Churchward, 1977
). We calculated the mean number of replication forks per cell, Fc, from the number of origins per cell (Table 2
) and the origin to terminus (O/T) ratio (Table 3
). All the dnaA(Sx) mutations reduced Fc significantly and irrespectively of the dnaX allele carried (Table 3
). For most mutations Fc was reduced to approximately 5060 % of the wild-type, but the dnaA893 mutation only reduced Fc to 75 % of the wild-type.
It can be concluded that the increased initiation mass observed for dnaA(Sx) cells was accompanied by a decrease in replication time. This indicates that replication forks in wild-type cells either do not travel with maximal velocity or make frequent pauses during replication of the chromosome. The decreased replication time subsequently resulted in a reduction of active replication forks per cell.
Increased gene dosage of datA suppresses the dnaX2016 Ts phenotype
The datA locus titrates large amounts of DnaA protein and an increased gene dosage of datA therefore delays initiation of DNA replication from oriC (Ogawa et al., 2002; Morigen et al., 2001
), induces asynchrony and decreases the replication time (C) (Morigen et al., 2003
). These phenotypes are similar to those observed for the dnaA(Sx) mutations isolated here. It was therefore tempting to speculate that the mechanism of suppression of the dnaX2016 mutation is related to one of these phenotypes.
Transformation of a dnaX2016 strain with either of the datA-containing plasmids pMOR8 (5 copies per cell; Morigen et al., 2001
) or pMOR6 (
10 copies per cell; Morigen et al., 2001
) increased the permissive temperature by 2 °C, whereas transformation with the parent plasmids pMW119 or pACYC177 did not change the permissive temperature (data not shown). The pMOR6 and pMOR8 plasmids also suppressed the SOS induction in a dnaX2016 mutant at 38 °C (Fig. 2
) and restored the plating efficiency (EOP) at 39 °C (Table 4
).
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DISCUSSION |
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SOS induction in dnaX2016 cells
The dnaX2016 mutation causes an immediate stop of DNA synthesis and gradual inhibition of cell division after temperature shift from 30 °C to 42 °C, whereas protein synthesis continues for at least 30 min (Chu et al., 1977). The SOS response was found to be somewhat induced even at permissive temperature in dnaX2016 cells. Because the dnaX2016 mutation affects the
and
clamp loader subunits of the replisome, the most likely explanation for this is that replication forks collapse with low frequency at permissive temperature, generating an SOS-inducing signal, but the collapsed forks can be efficiently restarted, and cells are fully viable (Seigneur et al., 1998
). At higher temperatures a strong SOS response was elicited in dnaX2016 cells, indicating that the SOS-inducing signal persisted or was continuously generated. An increased frequency of replication fork collapse that exceeds the cells' capacity for replication fork restart is the most likely explanation for the increased SOS induction. At high temperature, cell division is consequently inhibited and viability lost. The presence of any dnaA(Sx) allele or one of the datA plasmids suppressed the SOS induction of dnaX2016 mutant cells. This suggests that the dnaA(Sx) mutations reduce replication fork collapse in dnaX2016 cells.
Chromosome replication in the dnaA(Sx) mutants
Transcription of the dnaA gene was derepressed in all dnaA(Sx) mutants compared to wild-type, indicating that the DnaA(Sx) proteins were somewhat deficient in autorepression. Efficient repression of dnaA transcription is dependent on binding of DnaA associated with either ATP or ADP to a single DnaA-box and subsequent cooperative DnaA-ATP binding to adjacent 6-mer regions, located between the dnaAp1 and p2 promoters. The DnaA(Sx) mutant proteins were therefore expected to fall into two groups: those with altered binding to the DnaA-box and those that affected nucleotide binding. Both of these deficiencies were also expected to affect replication initiation from oriC, where binding of DnaA protein to DnaA-boxes and subsequently to DnaA-ATP-specific sites ensures open complex formation. In agreement with this it was found that all dnaA(Sx) mutations led to initiation at increased cell mass per origin (initiation mass; Table 2). In all cases, the increased initiation mass was accompanied by a decrease in the chromosome replication time (C-period). This is in agreement with previous observations where a decrease in availability of wild-type DnaA protein led to increased initiation mass and decreased C-period (Morigen et al., 2003
). A decreased C-period has also been observed in cells carrying dnaA mutations (Boye et al., 1996
; Morigen et al., 2003
) or a mutation in the hns gene (Atlung & Hansen, 2002
). An increased replication rate might therefore be a general consequence of reduced initiation frequency that in turn leads to fewer replication forks per cell mass. In wild-type cells activity of DNA polymerase III may therefore normally be limited by availability of nucleotides or other factors. Alternatively, frequent pausing of polymerase III could occur in wild-type cells, and a reduced concentration of active replication forks alleviates this pausing.
The synchrony of initiation with the single cell was also reduced by the dnaA(Sx) mutations, albeit to different extents. This reduced affinity of DnaA(Sx) proteins for the origin DnaA-boxes most likely led to an extended initiation interval in these cells. The extended initiation interval causes asynchrony because timely once-per-cell-cycle initiation (synchrony) is dependent on a period of initiation significantly shorter than the sequestration period (Skarstad & Løbner-Olesen, 2003) and the latter is not expected to be affected by dnaA(Sx) mutations. One group of mutants (dnaA893, dnaA895 and dnaA1111) initiate DNA replication very asynchronously and another group (dnaA1112, dnaA1113, dnaA1114 and dnaA1117) are only weakly asynchronous (Fig. 4
). The amino acid changes in the two groups of synchrony mutants are not clustered to a particular region of the DnaA protein (Fig. 1
), but connections to a particular property of the DnaA protein can be made (discussed below).
The highly asynchronous dnaA(Sx) mutants
Domains IIIa and IIIb of the DnaA protein comprise an AAA+ nucleotide-binding fold (Neuwald et al., 1999). The structure of domains III and IV was solved for the Aquifex aeolicus DnaA protein complexed with ADP (Erzberger et al., 2002
) and the structure of domain IV complexed with a DnaA-box was solved for E. coli DnaA (Fujikawa et al., 2003
). Fig. 5
shows the predicted structure of domains III+IV of the E. coli DnaA protein complexed with a DnaA-box. The positions of individual mutations are indicated.
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The dnaA1111 (S268T) mutation is located in the sensor I motif in 4, very close to where the
-phosphate would be if ATP was bound (Erzberger et al., 2002
). A changed sensing of the bound nucleotide is therefore the suggested cause of the reduced initiation capacity.
The dnaA893 (I379M) mutation is located in -helix
12 (Erzberger et al., 2002
), but outside the amphipathic part of this helix. This mutant deviated from the other dnaA(Sx) mutants by having only slightly increased initiation mass and replication rate. The present data show that the DnaA893 protein is very unstable (Fig. 3
), and the DnaA protein concentration is much lower than in wild-type cells. The DnaA893 protein therefore must be more active than the wild-type DnaA protein in DNA replication and autorepression. The increased activity of the DnaA893 protein suggests that this mutation eliminates a negative element; perhaps it escapes the RIDA-dependent inactivation.
The asynchrony of dnaA73, dnaA895 and dnaA1111 together with the classical dnaA mutants in the ATP-binding site (dnaA5, dnaA46, dnaA601 and dnaA604) (Skarstad et al., 1988) could indicate that asynchrony is a common property of mutations affecting binding, hydrolysis or sensing of the ATP molecule.
The slightly asynchronous dnaA(Sx) mutants
Four dnaA(Sx) mutations were only slightly affected in initiation synchrony. Two of these, dnaA1112 (T291N) located in the 5 strand (Erzberger et al., 2002
), and dnaA1113 (F250C) located in the
6 helix (Erzberger et al., 2002
), are in regions for which no specific function is known (Fig. 5
). F250 is however a very conserved amino acid and close to H252, which is changed to Y in dnaA46.
The dnaA1114 (Q370K) mutation is located in the amphipathic -helix 12, which in the model in Fig. 5
is broken by a random structure. This region is associated with phospholipids involved in rejuvenation of DnaA-ATP from DnaA-ADP (Garner et al., 1998
) and consequently the dnaA1114 mutation may alter the balance between these two species of DnaA protein to favour the DnaA-ADP form, inactive for both autorepression and initiation.
The dnaA1117 (R401S) mutation is located in the basic loop (Fig. 5; Erzberger et al., 2002
) and is in contact with the backbone phosphate of DNA via a water molecule during DnaA-box binding (Fujikawa et al., 2003
). This suggests that the dnaA1117 mutation leads to a weakening of DnaA-box binding, but maintains the specificity for DnaA-boxes. This is different from the base-specific binding of T435, changed to K in the asynchronous dnaA71 mutant (data not shown), which leads to loss of specificity in DNA binding (Blaesing et al., 2000
). This suggests an explanation for the clear differences in their effect on DNA replication in vivo.
The suppression mechanism is indirect
At least two different scenarios by which mutations in the dnaA gene suppress the dnaX2016 temperature sensitivity can be imagined. The DnaA [and DnaA(Sx)] proteins may contact the and/or
components of the DNA polymerase III holoenzyme either directly or indirectly. The suppressors obtained could be considered allele-specific suppressors of the dnaX2016 mutation. Because the affected amino acids in the mutant DnaA(Sx) proteins are located in both domains III and IV of DnaA (Fig. 1
), and affect different functions of the protein, we consider the DnaA and
and/or
interaction unlikely and therefore we investigated indirect suppression mechanisms.
All the dnaA(Sx) mutants analysed shared three characteristics: first, the SOS induction in dnaX2016 was suppressed at elevated temperature; second, initiation of replication was delayed in the cell cycle and occurred at an increased initiation mass; and third, replication of the chromosome was faster than in wild-type cells (the C-period was shorter). The presence of plasmids carrying the datA locus also delayed initiation of DNA replication in the cell cycle, and increased the replication rate and suppressed the dnaX2016 mutation. Delayed initiation in the cell cycle and an increased chromosomal replication rate both contribute to a reduced number of replication forks in cells. It is conceivable that the number of replication forks that collapse in the dnaX2016 cells also carrying a dnaA(Sx) suppressor is reduced at nonpermissive temperature as well. We speculate that the frequency of replication fork collapse in suppressed cells is such that they can be efficiently repaired by the host. This explains why the dnaA(Sx) mutations suppress both temperature sensitivity and SOS induction in cells carrying dnaX2016. Recently, support for an initiation deficiency model was obtained by Blinkova et al. (2003), who analysed the classical dnaA(Cs,Sx) mutants, primarily dnaA721. Like the dnaA(Sx) mutants described here, dnaA721 cells had increased length and reduced number of origins per cell. Suppression of the dnaX2016 temperature-sensitive phenotype by multicopy plasmids carrying the datA locus (this work) or by certain oriC mutations (Blinkova et al., 2003
) also supports an initiation deficiency model; in both cases binding of DnaA+ protein to oriC is reduced, leading to delayed initiation and an increase in the initiation mass.
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ACKNOWLEDGEMENTS |
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Received 16 September 2004;
revised 15 November 2004;
accepted 17 November 2004.
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