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Cellular localisation of the clamp protein during DNA replication
FEMS Microbiology Letters 216 (2002) 255^262
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Cellular localisation of the clamp protein during DNA replication Kritaya Kongsuwan , Brian P. Dalrymple, Gene Wij¡els, Phillip A. Jennings
1
CSIRO Division of Livestock Industries, Long Pocket Laboratories, 120 Meiers Road, Indooroopilly, Qld 4068, Australia Received 24 June 2002; received in revised form 5 September 2002; accepted 23 September 2002 First published online 12 October 2002
Abstract The L subunit of Escherichia coli DNA polymerase III holoenzyme was fused to the green fluorescent protein GFP. The gene fusion under the control of the heterologous lac promoter was used to replace the wild-type allele in the chromosome. The formation of GFP-L fluorescent foci in GFP-L expressing cells required DNA replication and their number per cell was dependent on cell growth. Examination of GFP-L foci in a synchronous round of replication suggested that DNA replication was accompanied by the recruitment of GFP-L foci near the midcell, followed by the rapid migration of the foci in opposite directions to the 1/4 and 3/4 positions during DNA replication. 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : DNA polymerase III; Beta subunit; DNA replication; Escherichia coli ; Green £uorescent protein ; Replication apparatus
1. Introduction The main function of the DNA polymerase III holoenzyme is the duplication of the Escherichia coli chromosome [1^3]. The holoenzyme synthesises DNA at approximately 500^1000 nucleotides per second [2,3]. The processivity of the enzyme is conferred by the sliding clamp L subunit protein [4,5]. The L clamp confers processivity to the core polymerase (KOa subunits) by directly binding to the catalytic K subunit of the core and tethering it to DNA [4^6]. L is a central component of the replication factory, playing a pivotal role in DNA replication, both encircling the DNA and being tethered to the DNA polymerase during replication. Its localisation during cell growth and division thus re£ects the location of the replication factory. Recent studies in Bacillus subtilis have shown that DNA replication occurs in stationary factories located at the centre of the cell and the DNA threads through a stationary replisome [7^9]. In support of this central replisome model in
* Corresponding author. Tel. : +61 (7) 3346 2512; Fax : +61 (7) 3346 2509. E-mail address : [email protected] (K. Kongsuwan). 1
Present address: Peptech Ltd, 1/35-41 Waterloo Road, North Ryde, NSW 1670, Australia.
E. coli is the observation that newly replicated DNA detected by pulse labelling with [3 H]thymidine is found near the midcell in most cells [10]. The stationary replication factory may be the common theme for bacterial DNA replication since many of the replication proteins, including L, are homologous and functionally equivalent in all bacteria. However, recent work using indirect immuno£uorescence microscopy [11] found that the L protein, which was ¢rst located at the midcell, migrates in opposite directions during replication. The result supports the translocating replication factories model in E. coli. In this model the paired replication apparatuses, which are ¢rst located close to each other at the middle of the cell, subsequently separate and migrate in opposite directions up to the 1/4 and 3/4 positions during DNA replication [12,13]. In this study, we have used fusion of the green £uorescent protein (GFP) to L to visualise the replication apparatuses with the aim of better understanding of the chromosome replication process during the cell cycle of E. coli. We show that GFP-L fusions form discrete foci in GFP-L expressing cells and that these foci are likely to represent the active sites of DNA replication. In addition, we ¢nd that the replisomes do not always reside in the centre of the cell, but move rapidly away towards the poles to occupy the 1/4 and 3/4 positions of the cell and remain there during most of DNA replication.
0378-1097 / 02 / $22.00 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 1 0 2 3 - 6
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XL1-Blue recA1 endA1 gyrA96 thi-1 hsdr17 supE44 relA1 lac [FP proAB lacIq ZvM15 Tn10 (Tetrr )] (Stratagene) was used for plasmid construction. PC2 strain leuB6, V3 , thyA47, rpsL153 (strR), deoC3, dnaT12 (ts), dnaC2 (ts) contains a temperature sensitive mutant of dnaT and dnaC genes. The dnaC gene encodes the DnaC protein involved in loading the DnaB helicase and the dnaT gene encodes DnaT protein which functions in primosome assembly. At the non-permissive temperature PC2 is blocked at the initiation stage of chromosome replication. DY329 W3110 vlacU169 nadA:Tn10 gal490 Vc1857v(crobioA) was used for gene replacement.
followed by 20 nt homologous to the kan cassette sequence (5P-gagagccacgatatcaaagaagatttttcaaatttaatcagaacattgtcatcgtaaacctTCTAGATTAGAAGAACTCGT-3P). The reverse recombinant primer was 92 nt long and contained a 5P segment that is complementary to the ¢rst 71 nt of the L gene excluding the start codon followed by a 21-nt 3P segment priming at the linker sequence and the 3P end of the GFPuv open reading frame (5P-ggacgaccacctaacggaccgctcacctgttgtagcggttttaataaatgctcacgttctacggtaaatttGGGCCCCGCGCCTTTGTAGAG-3P). The PCR fragment was gel puri¢ed and introduced into E. coli DY329 by electroporation (Bio-Rad Gene Pulser set at 1.8 kV, 25 WF with Pulse controller of 200 6). Correct insertion into the chromosome of the kan-placGFP fragment was veri¢ed by diagnostic PCR with appropriate primers (see Fig. 1B).
2.2. Plasmid constructions
2.4. Isolation of PC2 GFP-L transductant
An in-frame fusion of the L coding sequence to the 3P end of the GFP gene was achieved by ligating a bluntended L polymerase chain reaction (PCR) fragment to SfrI-digested placGFP to create placGFP-L. The placGFP plasmid was obtained by inserting lac promoter upstream of the gfpuv gene in pGFPuv (Clontech, Palo Alto, CA, USA). The primers used to amplify L gene directly from XL1-Blue genomic DNA were 5P-AAATTTACCGTAGAACGTGAG-3P and 5P-ACATTACAGTCTCATTGGCAT-3P.
Transductant of the gene encoding the GFP-L fusion protein was obtained by using phage P1 (C600) (a gift from Dr Nick Dixon, ANU) with DY329 L 6 s GFP-L as the donor strain and PC2 (temperature sensitive mutant dnaC2 mutant) as the recipient strain. Transductants were selected on kanamycin plates following incubation at 30‡C.
2. Materials and methods 2.1. E. coli strains
2.3. Chromosome integration of kan-placGFP cassette The kanamycin resistant cassette (kan) was ampli¢ed from pCR 2.1-TOPO (Invitrogen) with primers 5P-GCATGCATCTAGATATGGACAGCAAGCGAACCG-3P and 5P-TGGGCCCTCTAGATTAGAAGAACTCGTCAAGAAGG-3P and cloned into placGFP-L to generate pkanlacGFP-L. For gene replacement, the fragment harbouring the kan selection marker, lac promoter (plac), gfp gene, linker sequence and sequence homologous to the integration sites on either end, was ampli¢ed from the plasmid pkan-lacGFP-L. The chimeric forward recombination primers were 82 nucleotides (nt) long and consisted of a 62-nt 5P segment homologous to a region directly upstream of the start ATG of the L gene (in lower case)
2.5. Immunoblotting Total proteins were prepared from cells grown in Luria^ Bertani (LB) medium at 30‡C and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose and the ¢lter was probed with rabbit polyclonal antibody to L followed by a horseradish peroxidase-coupled secondary antibody (Silenus). Proteins were visualised with 4-chloro-1-naphthol as substrate. 2.6. Measurement of DNA synthesis The rate of DNA synthesis was determined at 10-min intervals by transferring 200 Wl of a growing culture of E. coli to a tube containing 1 WCi [3 H]thymidine (speci¢c activity of 15^30 Ci mmol31 ) (Amersham Biosciences). After incubation at 37‡C for 2 min, the incorporation of
C Fig. 1. Replacement of L with an insertion allele kan placGFP-L. A: A strategy for generating gene replacement (modi¢ed from [14]). Shaded rectangles represent sequences in the recombinant primers that are identical to sequences at either side of the start of the L gene. Arrowheads are sequences in the primers that are homologous to the ends of the kan-placGFP cassette to be introduced. The cassette is generated by PCR and introduced into E. coli DY329, which was induced for Exo, Beta and Gam functions that promote recombination. Recombination occurs between the homologous sequences on the linear cassette and the target replacing the target segment with the cassette. The small arrows marked with capital letters are the PCR primer sites used to verify the gene replacement. The predicted size of the PCR products for the insertion allele and wild-type are shown with the latter in parentheses. B: Diagnostic PCR to verify the replacement of L with the insertion allele. The gel shows the PCR products using primer pairs AB (lanes 1 and 2), CD (lanes 3 and 4) and EF (lanes 5 and 6). Templates are genomic DNA from DY329 wild-type (lanes 2, 4 and 6) and DY329 L 6 s GFP-L (lanes 1, 3 and 5). The sizes of DNA markers are shown in kilobases. C: Immunoblots of total proteins from wild-type DY329 (lane 1) and L 6 s GFP-L (lane 2) cells probed with polyclonal antibody to L.
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radioactivity was terminated by the addition of 1 ml of 10% trichloroacetic acid. Acid-insoluble materials were processed and counted on glass ¢lters.
3. Results and discussion
2.7. Fluorescence microscopy and measurement
Our initial attempts to place the fusion gene in the chromosome, replacing the wild-type gene, i.e. downstream of dnaA and under control of the dnaA regulon, were unsuccessful, suggesting that GFP-L expression at the wild-type level is insu⁄cient to enable cell viability. To overcome this problem, GFP-L driven by the lac promoter was used to replace the native L. Fig. 1A illustrates the oligonucleotide-directed gene replacement system [14] to replace L with GFP-L in the chromosome. To verify correct integration and the integrity of the fusion gene, genomic DNA prepared from kanamycin resistant transformants was used as template in PCR reactions with three pairs of diagnostic primers (Fig. 1B). GFP gene speci¢c primers (C and D), primer A is homologous to the kanamycin resistant gene and primer B is complementary to the 5P end of L. Primer E is from 400 bases upstream of the start of the L gene, while primer F is complementary to
Strains were grown in LB or M9/0.5% glucose medium supplemented with kanamycin (50 Wg ml31 ) at 30‡C. For live cell observations, a suspension of living cells was dropped on to a coverglass that was coated with poly-Llysine, and mounted on a slide with 0.5% low melting point agarose containing medium. For better viewing, some cells were ¢xed with methanol (which did not appear to a¡ect GFP signal or localisation of the fusion protein). Microscopy was performed on a Leica TCS 4D or a BioRad multiphoton confocal microscope (100U/1.4 lens). To randomise the sampling, all cells within a ¢eld, except those in aggregates, were analysed. For measurements of cell length, position and £uorescence intensity of foci, Image J and Adobe Photoshop 6.0 software were used.
3.1. Replacement of L with GFP-L in the chromosome
Fig. 2. E¡ect of nalidixic acid on GFP-L signals. DY329 L 6 s GFP-L cells grown in LB were treated with DNA replication blocker nalidixic acid and samples were taken for microscopy and DNA synthesis measurement. For microscopy, cells were ¢xed in 70% methanol for 5 min. A: Untreated cells. B: Cells treated with nalidixic acid for 15 min. The scale bar represents 1 Wm. C: Measurements of DNA replication in untreated cells and in nalidixic acid treated cells. Squares indicate untreated control and triangles indicate nalidixic acid treated cells. DNA synthesis is expressed as a percentage of the untreated control at time zero.
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Measurements of DNA synthesis showed that treatment of nalidixic acid resulted in substantial reduction of DNA synthesis within 10 min after treatment (Fig. 2C). The GFP-L foci were observed in 6 3% of the cells (total 644 cells counted) 15 min after the addition of nalidixic acid (Fig. 2B), compared to the untreated control with 75% of cells with one or more foci (756 cells counted) (Fig. 2A). The result supports the argument that the presence of GFP-L foci is associated with ongoing DNA replication. 3.3. The number of GFP-L foci is dependent on growth conditions and phases
Fig. 3. Analysis of the number of GFP-L foci per cell in GFP-L expressing cells. DY329 L 6 s GFP-L cells were grown overnight in LB medium with 50 Wg ml31 kanamycin at 30‡C, diluted in fresh M9/0.5% glucose or LB and grown to early exponential phase or stationary phase before harvesting. Histograms show the percentage of cells containing £uorescent GFP-L foci. At least 250 cells were counted for each class.
the sequence at the 3P end of the L gene. Two colonies displayed PCR products consistent with the predicted sizes, indicative of the insertion at the 5P end of L (Fig. 1B). Furthermore, correct insertion was con¢rmed by sequencing of the 3.2-kb PCR product obtained by using primers E and F (Fig. 1B, lane 5). Immunoblot analysis of total proteins from wild-type and L 6 s GFP-L recombinant con¢rms the presence of a hybrid protein replacing the wild-type protein (Fig. 1C). The amount of the hybrid fusion protein detected was estimated assuming immunoblot to be about twice that of the wild-type L (Fig. 1C), which has previously been estimated to be about 300^350 molecules per cell [15].
To determine whether GFP-L foci localisation is in£uenced by growth rate, we examined the number of foci per cell in the GFP-L strain grown in minimal medium (M9/ 0.5% glucose) and in LB at 30‡C. We also examined the e¡ect of growth phase on number of foci by analyzing cultures that had entered stationary phase (OD600 = 2.2). Although 79% of the exponential phase cells grown in rich medium contained one or more foci, only 26% contained one or two foci after entering stationary phase (Fig. 3). In cells growing in M9 medium, 39% of cells had no foci compared to 21% in LB medium. Taken together, the results suggest growth conditions substantially a¡ect the presence and the number of GFP-L foci per cell, consistent with GFP-L foci representing active replisomes. 3.4. Subcellular localisation of the GFP-L during the cell cycle Expression of discrete GFP-L foci in cells allows visualisation of the replisomes and hence their localisation in the
3.2. Formation of GFP-L foci requires DNA replication When the strain harbouring GFP-L (DY329 L 6 s GFP-L) was grown in a rich medium (LB) and prepared for live cell £uorescence microscopy, cytoplasmic £uorescence could be detected in all cells and most cells (75%) showed one or more discrete £uorescent foci (Fig. 2A). As a control, the wild-type strain (DY329) did not show any £uorescence and GFP encoded by the pGFPuv plasmid was dispersed throughout the cells (data not shown). It is likely that GFP-L foci correspond to the localisation of active L involved in DNA replication. To determine whether this is the case, we treated exponentially growing cells with 200 Wg ml31 nalidixic acid (Sigma-Aldrich) and examined cells 15 min later for GFP-L foci. Nalidixic acid is an inhibitor of DNA gyrase and topoisomerase IV, enzymes required for generating negative supercoils and unlinking DNA during replication.
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Fig. 4. Fluorescent micrographs of non-synchronised GFP-L expressing cells. DY329 L 6 s GFP-L cells were grown overnight in LB medium with 50 Wg ml31 kanamycin at 30‡C, diluted 200-fold in fresh LB, and grown to early exponential phase. At this point, cells were removed and examined by confocal microscopy. Representative images of cells containing one (A,B), two (C^F), three (G^J) and four (K,L) foci are shown. Scale bar, 2 Wm.
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cells can be studied. To obtain the data on the positions of the replisomes during the cell cycle of E. coli, we ¢rst examined the relative positions of GFP-L foci within cells undergoing exponential growth (OD600 = 0.6). Fig. 4 shows typical examples of £uorescence micrographs of GFP-L expressing cells grown in LB medium and the analysis of their intracellular locations is shown in Fig. 5. Shorter cells contained a single focus that was preferentially found near the cell midpoint (Figs. 4A,B, and 5A). Two foci were seen on either side of the midcell, often appeared to be symmetrically displaced from the centre and were often clustered at 1/4 and 3/4 positions (Figs. 4C^F and 5B). Three-focus cells predominantly had two foci located close together near the 1/4 position with the third focus at the 3/4 position (Figs. 4G^I and 5C). Cells containing four foci comprised only 4% of the population, and they usually appeared to have two pairs, where each pair was spaced similarly around the 1/4 and 3/4 positions (Figs. 4J^L and 5D). The number of foci per cell showed a strong correlation with cell length, which in E. coli is an approximate indicator of progression through the cell cycle [16]. The average lengths for cells containing one, two, three, and four foci were 1.98 U 0.34, 2.62 U 0.49, 3.43 U 0.57 and 3.73 U 0.58 Wm, respectively, which were all signi¢cantly di¡erent from each other (P 6 0.001; multiple range test). Despite the exponential growth in rich growth medium, many DY329 L 6 s GFP cells contained no visible foci and most cells had one or two foci (Fig. 3). The reason for this is not clear. Our data showed that the GFP-L expressing strain grew more slowly than the wild-type strain (DY329) having a 55-min doubling time compared to the 35-min doubling time of the wild-type when both strains were grown in LB at 30‡C (data not shown). These results suggest that cells without foci had non-replicating nucleoid and that multiple fork formation was rare. Alternatively we could not detect faint foci due to the high background of di¡used GFP-L £uorescence in the cytoplasm. To further examine the relationship between the cellular location of GFP-L and the time of DNA replication we Table 1 Analysis of GFP-L foci in cells synchronised for initiation of chromosome replication Time after 30‡C shifta (min)
Fig. 5. Localisation of GFP-L foci in non-synchronised cells. The same population as shown in Fig. 3 was used in the analysis but cells were ¢xed in 70% methanol before microscopy. Cell lengths and positions of £uorescent signals were measured and the results plotted. Each dot/triangle represents a GFP-L focus. Distribution patterns for A: one focus (88 cells); B: two foci (119 cells); C: three foci (52 cells); D: four foci (18 cells).
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c
0 (548) 10 (368) 25 (382) 40 (250) 55 (373)
Percentage of cells with n focib 0
1
2
3
4
99 28 14 53 100
1 65 5 13 61
^ 7 72 29 ^
^ ^ 61 2 ^
^ ^ 9 3 ^
a Cultures of PC2/GFP-L were subjected to three temperature shifts as described in the text. b The data are cumulative from two independent experiments, each of which gave similar results. c Total cells counted.
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Fig. 6. Intracellular localisation of GFP-L foci in cells synchronised for initiation of chromosome replication. PC2/GFP-L cells grown at 30‡C in LB plus thymine (50 Wg ml31 ) were shifted to the non-permissive temperature of 42‡C for 1 h to stop DNA replication initiation. To synchronously initiate DNA replication, the cells were shifted to 30‡C for 6 min before being transferred again to 42‡C to inhibit further initiation. A: The rate of DNA synthesis in GFP-L/PC2 cells at 42‡C after a short temperature shift. The average of three replicates is shown. B: Fluorescence images of cells progressing through the cell cycle at 42‡C after temperature shift. Times after the 30‡C temperature shift are indicated. Scale bar, 1 Wm.
analysed the number (Table 1) and location of GFP-L foci in cells synchronised for initiation of chromosome replication. To do this, we constructed GFP-L transductant derived from a temperature sensitive dnaC2 mutant (strain PC2). The DnaC protein forms a complex with DnaB (helicase) and helps load DnaB onto DNA. At the nonpermissive temperature (42‡C) the PC2 strain is inactive for replication initiation, but replication initiation can be restored upon transfer to 30‡C [11,12]. A culture of PC2/ GFP-L strain in exponential phase at 30‡C was transferred to 42‡C (to allow completion of current rounds of replication and synchronise cells), then transferred to 30‡C for 6 min (to allow a single round of replication to be initi-
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ated) before being shifted back to 42‡C. Before transfer to 30‡C, GFP-L was dispersed throughout the cells and there were no apparent foci in most cells (Fig. 6B). However, within 10 min after the temperature shift, rather fuzzy £uorescent GFP-L foci were observed in 72% of cells (Table 1) and these were usually recruited near midcell in short cells and near the quarter positions in longer, twofocus cells (Fig. 6B, 10 min, arrows). At 25 min, most (86%) cells had very bright, well-de¢ned foci of GFP-L. When there were two foci, these were located at cell quarter positions. Longer cells appeared to have two pairs of foci, where each pair was spaced similarly around the 1/4 and 3/4 positions (Fig. 6B). At 40 min, GFP-L foci were
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observed in 47% of cells and these foci had similar distribution pattern to those observed at 25 min, but the foci were less bright, more di¡use and in some cells the foci were starting to disappear (Fig. 6B, 40 min, arrow). At 55 min the £uorescence was dispersed throughout the cell (Fig. 6B) and rarely formed discrete foci. The peaks of DNA synthesis (Fig. 6A) are correlated with bright GFP-L clusters localised at cell quarter positions (Fig. 6B, 25 min). From these results we conclude that immediately following DNA replication initiation the replication machinery was ¢rst recruited near midcell position (or future midcell in longer cells). As DNA replication progresses the replication machinery splits and the submachines rapidly move apart to occupy the cell quarter positions and reside there for the duration of DNA replication. The fractions of cells with two foci at 10 min and those with four foci at 25 min (7% and 9% respectively, Table 1) are consistent with the interpretation that cells with two foci convert to cells with four foci. Because only one round of DNA replication occurred in these cells, the apparently moved foci cannot be re-initiation events. Rapid photobleaching of the GFP-L has made it impossible for us to carry out time-lapse studies to directly con¢rm that the GFP-L foci ¢rst form at the centre, separate and then move away. Our more direct detection of the replisome by use of GFP-L fusion o¡ers new insights into the behaviour of the replisomes in living cells. The replication machinery in E. coli does not remain in the centre of the cell until the terminus has been replicated as suggested by the stationary replisome model [7,8,17,18]. The experimental results are consistent with the translocating replication factories model [11^13]. Using indirect immuno£uorescence in ¢xed cells, it has recently been found that in the absence of DNA replication, L molecules were localised at or near the cell poles [11]. We did not observe the clustering of inactive L at cell poles, but rather the £uorescence was dispersed throughout the cells. This may re£ect the di¡erent methods used in the two studies. Alternatively, the overall high expression of GFP-L expressed from the strong lac promoter in our cells (Fig. 1C) may mask any aggregation of L molecules in the polar region. The mechanism that allows the replication machinery to migrate is not known, but the movement seems to be rapid (‘jumping’) in opposite directions to 1/4 and 3/4 positions during chromosome replication. This migration may be mediated by the chromosome segregation and/or by the inherent nature of cell division to generate a nucleoidfree zone at the cell centre for septum formation [19].
Acknowledgements We thank Drs Ross Tellam, Rob Moore and Professor John Mattick for critical reading of the manuscript and Professor Sota Hiraga for his suggestion to use E. coli PC2
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to synchronise for initiation of chromosome replication. This work was funded in association with a Corporate Fellowship to P.A.J. within a CSIRO Chief Executive Special Project, the Bioactive Molecules Initiative.
References [1] Kornberg, A. and Baker, T. (1992) in : DNA Replication. W.H. Freeman and Company, New York. [2] Kelman, Z. and O’Donnell, M. (1995) DNA polymerase III holoenzyme: structure and function of a chromosomal replicating machine. Annu. Rev. Biochem. 64, 171^200. [3] Baker, T.A. and Bell, S.P. (1998) Polymerases and the replisome: machines within machines. Cell 92, 295^305. [4] LaDuca, R.J., Crute, J.J., McHenry, C.S. and Bambara, R.A. (1986) The L subunit of the Escherichia coli DNA polymerase III holoenzyme interacts functionally with the catalytic core in the absence of other subunits. J. Biol. Chem. 26, 7550^7557. [5] Stukenberg, P.T., Studwell-Vaughan, P.S. and O’Donnell, M. (1991) Mechanism of the sliding L-clamp of DNA polymerase III holoenzyme. J. Biol. Chem. 266, 11328^11334. [6] Kim, D.R. and McHenry, C.S. (1996) Identi¢cation of the L-binding domain of the K subunit of Escherichia coli polymerase III holoenzyme. J. Biol. Chem. 271, 20699^20704. [7] Lemon, K.P. and Grossman, A.D. (1998) Localization of bacterial DNA polymerase : evidence for a factory mode of replication. Science 282, 1516^1519. [8] Lemon, K.P. and Grossman, A.D. (2000) Movement of replicating DNA through a stationary replisome. Mol. Cell 6, 1321^1330. [9] Sawitzke, J. and Austin, S. (2001) An analysis of the factory model for chromosome replication and segregation in bacteria. Mol. Microbiol. 40, 786^794. [10] Koppes, L.J., Woldringh, C.L. and Nanninga, N. (1999) Escherichia coli contains a DNA replication compartment in the cell center. Biochimie 81, 803^810. [11] Onogi, T., Ohsumi, K., Katayama, T. and Hiraga, S. (2002) Replication-dependent recruitment of the L-subunit of DNA polymerase III from cytosolic spaces to replication fork in Escherichia coli. J. Bacteriol. 184, 867^870. [12] Hiraga, S., Ichinose, C., Onogi, T., Niki, H. and Yamazoe, M. (2000) Bi-directional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli. Genes Cells 5, 327^ 341. [13] Hiraga, S. (2000) Dynamic localization of bacterial and plasmid chromosomes. Annu. Rev. Genet. 34, 21^59. [14] Yu, D., Ellis, H., Lee, E.-C., Jenkins, N.A., Copeland, N.G. and Court, D.L. (2000) An e⁄cient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5978^5983. [15] Leu, F.P., Hingorani, M.M., Turner, J. and O’Donnell, M. (2000) The N subunit of DNA polymerase serves as the sliding clamp unloader in E. coli. J. Biol. Chem. 275, 34609^34618. [16] Koppes, L.J.H., Woldringh, C.L.W. and Nanninga, N. (1978) Size variations and correlation of di¡erent cell cycle events in slow-growing Escherichia coli. J. Bacteriol. 134, 423^433. [17] Roos, M., van Geel, A.B.M., Aarsman, M.E., Veuskens, J.T., Woldringh, C.L. and Nanninga, N. (1999) Localisation of oriC during the cell cycle of Eschericia coli as analysed by £uorescent in situ hybridisation. Biochimie 81, 797^802. [18] Imai, Y., Ogasawara, N., Ishigo-Oka, D., Kadoya, R., Daito, T. and Moriya, S. (2000) Subcellular localization of Dna-initiation proteins of Bacillus subtilis : evidence that chromosome replication begins at either edge of the nucleoids. Mol. Microbiol. 36, 1037^1048. [19] Chaudhuri, D.R., Gordon, G.S. and Wright, A. (2000) How does a bacterium ¢nd its middle? Nature Struct. Biol. 7, 997^999.
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Cellular localisation of the clamp protein during DNA replication Kritaya Kongsuwan , Brian P. Dalrymple, Gene Wij¡els, Phillip A. Jennings
1
CSIRO Division of Livestock Industries, Long Pocket Laboratories, 120 Meiers Road, Indooroopilly, Qld 4068, Australia Received 24 June 2002; received in revised form 5 September 2002; accepted 23 September 2002 First published online 12 October 2002
Abstract The L subunit of Escherichia coli DNA polymerase III holoenzyme was fused to the green fluorescent protein GFP. The gene fusion under the control of the heterologous lac promoter was used to replace the wild-type allele in the chromosome. The formation of GFP-L fluorescent foci in GFP-L expressing cells required DNA replication and their number per cell was dependent on cell growth. Examination of GFP-L foci in a synchronous round of replication suggested that DNA replication was accompanied by the recruitment of GFP-L foci near the midcell, followed by the rapid migration of the foci in opposite directions to the 1/4 and 3/4 positions during DNA replication. 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : DNA polymerase III; Beta subunit; DNA replication; Escherichia coli ; Green £uorescent protein ; Replication apparatus
1. Introduction The main function of the DNA polymerase III holoenzyme is the duplication of the Escherichia coli chromosome [1^3]. The holoenzyme synthesises DNA at approximately 500^1000 nucleotides per second [2,3]. The processivity of the enzyme is conferred by the sliding clamp L subunit protein [4,5]. The L clamp confers processivity to the core polymerase (KOa subunits) by directly binding to the catalytic K subunit of the core and tethering it to DNA [4^6]. L is a central component of the replication factory, playing a pivotal role in DNA replication, both encircling the DNA and being tethered to the DNA polymerase during replication. Its localisation during cell growth and division thus re£ects the location of the replication factory. Recent studies in Bacillus subtilis have shown that DNA replication occurs in stationary factories located at the centre of the cell and the DNA threads through a stationary replisome [7^9]. In support of this central replisome model in
* Corresponding author. Tel. : +61 (7) 3346 2512; Fax : +61 (7) 3346 2509. E-mail address : [email protected] (K. Kongsuwan). 1
Present address: Peptech Ltd, 1/35-41 Waterloo Road, North Ryde, NSW 1670, Australia.
E. coli is the observation that newly replicated DNA detected by pulse labelling with [3 H]thymidine is found near the midcell in most cells [10]. The stationary replication factory may be the common theme for bacterial DNA replication since many of the replication proteins, including L, are homologous and functionally equivalent in all bacteria. However, recent work using indirect immuno£uorescence microscopy [11] found that the L protein, which was ¢rst located at the midcell, migrates in opposite directions during replication. The result supports the translocating replication factories model in E. coli. In this model the paired replication apparatuses, which are ¢rst located close to each other at the middle of the cell, subsequently separate and migrate in opposite directions up to the 1/4 and 3/4 positions during DNA replication [12,13]. In this study, we have used fusion of the green £uorescent protein (GFP) to L to visualise the replication apparatuses with the aim of better understanding of the chromosome replication process during the cell cycle of E. coli. We show that GFP-L fusions form discrete foci in GFP-L expressing cells and that these foci are likely to represent the active sites of DNA replication. In addition, we ¢nd that the replisomes do not always reside in the centre of the cell, but move rapidly away towards the poles to occupy the 1/4 and 3/4 positions of the cell and remain there during most of DNA replication.
0378-1097 / 02 / $22.00 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 1 0 2 3 - 6
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XL1-Blue recA1 endA1 gyrA96 thi-1 hsdr17 supE44 relA1 lac [FP proAB lacIq ZvM15 Tn10 (Tetrr )] (Stratagene) was used for plasmid construction. PC2 strain leuB6, V3 , thyA47, rpsL153 (strR), deoC3, dnaT12 (ts), dnaC2 (ts) contains a temperature sensitive mutant of dnaT and dnaC genes. The dnaC gene encodes the DnaC protein involved in loading the DnaB helicase and the dnaT gene encodes DnaT protein which functions in primosome assembly. At the non-permissive temperature PC2 is blocked at the initiation stage of chromosome replication. DY329 W3110 vlacU169 nadA:Tn10 gal490 Vc1857v(crobioA) was used for gene replacement.
followed by 20 nt homologous to the kan cassette sequence (5P-gagagccacgatatcaaagaagatttttcaaatttaatcagaacattgtcatcgtaaacctTCTAGATTAGAAGAACTCGT-3P). The reverse recombinant primer was 92 nt long and contained a 5P segment that is complementary to the ¢rst 71 nt of the L gene excluding the start codon followed by a 21-nt 3P segment priming at the linker sequence and the 3P end of the GFPuv open reading frame (5P-ggacgaccacctaacggaccgctcacctgttgtagcggttttaataaatgctcacgttctacggtaaatttGGGCCCCGCGCCTTTGTAGAG-3P). The PCR fragment was gel puri¢ed and introduced into E. coli DY329 by electroporation (Bio-Rad Gene Pulser set at 1.8 kV, 25 WF with Pulse controller of 200 6). Correct insertion into the chromosome of the kan-placGFP fragment was veri¢ed by diagnostic PCR with appropriate primers (see Fig. 1B).
2.2. Plasmid constructions
2.4. Isolation of PC2 GFP-L transductant
An in-frame fusion of the L coding sequence to the 3P end of the GFP gene was achieved by ligating a bluntended L polymerase chain reaction (PCR) fragment to SfrI-digested placGFP to create placGFP-L. The placGFP plasmid was obtained by inserting lac promoter upstream of the gfpuv gene in pGFPuv (Clontech, Palo Alto, CA, USA). The primers used to amplify L gene directly from XL1-Blue genomic DNA were 5P-AAATTTACCGTAGAACGTGAG-3P and 5P-ACATTACAGTCTCATTGGCAT-3P.
Transductant of the gene encoding the GFP-L fusion protein was obtained by using phage P1 (C600) (a gift from Dr Nick Dixon, ANU) with DY329 L 6 s GFP-L as the donor strain and PC2 (temperature sensitive mutant dnaC2 mutant) as the recipient strain. Transductants were selected on kanamycin plates following incubation at 30‡C.
2. Materials and methods 2.1. E. coli strains
2.3. Chromosome integration of kan-placGFP cassette The kanamycin resistant cassette (kan) was ampli¢ed from pCR 2.1-TOPO (Invitrogen) with primers 5P-GCATGCATCTAGATATGGACAGCAAGCGAACCG-3P and 5P-TGGGCCCTCTAGATTAGAAGAACTCGTCAAGAAGG-3P and cloned into placGFP-L to generate pkanlacGFP-L. For gene replacement, the fragment harbouring the kan selection marker, lac promoter (plac), gfp gene, linker sequence and sequence homologous to the integration sites on either end, was ampli¢ed from the plasmid pkan-lacGFP-L. The chimeric forward recombination primers were 82 nucleotides (nt) long and consisted of a 62-nt 5P segment homologous to a region directly upstream of the start ATG of the L gene (in lower case)
2.5. Immunoblotting Total proteins were prepared from cells grown in Luria^ Bertani (LB) medium at 30‡C and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose and the ¢lter was probed with rabbit polyclonal antibody to L followed by a horseradish peroxidase-coupled secondary antibody (Silenus). Proteins were visualised with 4-chloro-1-naphthol as substrate. 2.6. Measurement of DNA synthesis The rate of DNA synthesis was determined at 10-min intervals by transferring 200 Wl of a growing culture of E. coli to a tube containing 1 WCi [3 H]thymidine (speci¢c activity of 15^30 Ci mmol31 ) (Amersham Biosciences). After incubation at 37‡C for 2 min, the incorporation of
C Fig. 1. Replacement of L with an insertion allele kan placGFP-L. A: A strategy for generating gene replacement (modi¢ed from [14]). Shaded rectangles represent sequences in the recombinant primers that are identical to sequences at either side of the start of the L gene. Arrowheads are sequences in the primers that are homologous to the ends of the kan-placGFP cassette to be introduced. The cassette is generated by PCR and introduced into E. coli DY329, which was induced for Exo, Beta and Gam functions that promote recombination. Recombination occurs between the homologous sequences on the linear cassette and the target replacing the target segment with the cassette. The small arrows marked with capital letters are the PCR primer sites used to verify the gene replacement. The predicted size of the PCR products for the insertion allele and wild-type are shown with the latter in parentheses. B: Diagnostic PCR to verify the replacement of L with the insertion allele. The gel shows the PCR products using primer pairs AB (lanes 1 and 2), CD (lanes 3 and 4) and EF (lanes 5 and 6). Templates are genomic DNA from DY329 wild-type (lanes 2, 4 and 6) and DY329 L 6 s GFP-L (lanes 1, 3 and 5). The sizes of DNA markers are shown in kilobases. C: Immunoblots of total proteins from wild-type DY329 (lane 1) and L 6 s GFP-L (lane 2) cells probed with polyclonal antibody to L.
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radioactivity was terminated by the addition of 1 ml of 10% trichloroacetic acid. Acid-insoluble materials were processed and counted on glass ¢lters.
3. Results and discussion
2.7. Fluorescence microscopy and measurement
Our initial attempts to place the fusion gene in the chromosome, replacing the wild-type gene, i.e. downstream of dnaA and under control of the dnaA regulon, were unsuccessful, suggesting that GFP-L expression at the wild-type level is insu⁄cient to enable cell viability. To overcome this problem, GFP-L driven by the lac promoter was used to replace the native L. Fig. 1A illustrates the oligonucleotide-directed gene replacement system [14] to replace L with GFP-L in the chromosome. To verify correct integration and the integrity of the fusion gene, genomic DNA prepared from kanamycin resistant transformants was used as template in PCR reactions with three pairs of diagnostic primers (Fig. 1B). GFP gene speci¢c primers (C and D), primer A is homologous to the kanamycin resistant gene and primer B is complementary to the 5P end of L. Primer E is from 400 bases upstream of the start of the L gene, while primer F is complementary to
Strains were grown in LB or M9/0.5% glucose medium supplemented with kanamycin (50 Wg ml31 ) at 30‡C. For live cell observations, a suspension of living cells was dropped on to a coverglass that was coated with poly-Llysine, and mounted on a slide with 0.5% low melting point agarose containing medium. For better viewing, some cells were ¢xed with methanol (which did not appear to a¡ect GFP signal or localisation of the fusion protein). Microscopy was performed on a Leica TCS 4D or a BioRad multiphoton confocal microscope (100U/1.4 lens). To randomise the sampling, all cells within a ¢eld, except those in aggregates, were analysed. For measurements of cell length, position and £uorescence intensity of foci, Image J and Adobe Photoshop 6.0 software were used.
3.1. Replacement of L with GFP-L in the chromosome
Fig. 2. E¡ect of nalidixic acid on GFP-L signals. DY329 L 6 s GFP-L cells grown in LB were treated with DNA replication blocker nalidixic acid and samples were taken for microscopy and DNA synthesis measurement. For microscopy, cells were ¢xed in 70% methanol for 5 min. A: Untreated cells. B: Cells treated with nalidixic acid for 15 min. The scale bar represents 1 Wm. C: Measurements of DNA replication in untreated cells and in nalidixic acid treated cells. Squares indicate untreated control and triangles indicate nalidixic acid treated cells. DNA synthesis is expressed as a percentage of the untreated control at time zero.
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Measurements of DNA synthesis showed that treatment of nalidixic acid resulted in substantial reduction of DNA synthesis within 10 min after treatment (Fig. 2C). The GFP-L foci were observed in 6 3% of the cells (total 644 cells counted) 15 min after the addition of nalidixic acid (Fig. 2B), compared to the untreated control with 75% of cells with one or more foci (756 cells counted) (Fig. 2A). The result supports the argument that the presence of GFP-L foci is associated with ongoing DNA replication. 3.3. The number of GFP-L foci is dependent on growth conditions and phases
Fig. 3. Analysis of the number of GFP-L foci per cell in GFP-L expressing cells. DY329 L 6 s GFP-L cells were grown overnight in LB medium with 50 Wg ml31 kanamycin at 30‡C, diluted in fresh M9/0.5% glucose or LB and grown to early exponential phase or stationary phase before harvesting. Histograms show the percentage of cells containing £uorescent GFP-L foci. At least 250 cells were counted for each class.
the sequence at the 3P end of the L gene. Two colonies displayed PCR products consistent with the predicted sizes, indicative of the insertion at the 5P end of L (Fig. 1B). Furthermore, correct insertion was con¢rmed by sequencing of the 3.2-kb PCR product obtained by using primers E and F (Fig. 1B, lane 5). Immunoblot analysis of total proteins from wild-type and L 6 s GFP-L recombinant con¢rms the presence of a hybrid protein replacing the wild-type protein (Fig. 1C). The amount of the hybrid fusion protein detected was estimated assuming immunoblot to be about twice that of the wild-type L (Fig. 1C), which has previously been estimated to be about 300^350 molecules per cell [15].
To determine whether GFP-L foci localisation is in£uenced by growth rate, we examined the number of foci per cell in the GFP-L strain grown in minimal medium (M9/ 0.5% glucose) and in LB at 30‡C. We also examined the e¡ect of growth phase on number of foci by analyzing cultures that had entered stationary phase (OD600 = 2.2). Although 79% of the exponential phase cells grown in rich medium contained one or more foci, only 26% contained one or two foci after entering stationary phase (Fig. 3). In cells growing in M9 medium, 39% of cells had no foci compared to 21% in LB medium. Taken together, the results suggest growth conditions substantially a¡ect the presence and the number of GFP-L foci per cell, consistent with GFP-L foci representing active replisomes. 3.4. Subcellular localisation of the GFP-L during the cell cycle Expression of discrete GFP-L foci in cells allows visualisation of the replisomes and hence their localisation in the
3.2. Formation of GFP-L foci requires DNA replication When the strain harbouring GFP-L (DY329 L 6 s GFP-L) was grown in a rich medium (LB) and prepared for live cell £uorescence microscopy, cytoplasmic £uorescence could be detected in all cells and most cells (75%) showed one or more discrete £uorescent foci (Fig. 2A). As a control, the wild-type strain (DY329) did not show any £uorescence and GFP encoded by the pGFPuv plasmid was dispersed throughout the cells (data not shown). It is likely that GFP-L foci correspond to the localisation of active L involved in DNA replication. To determine whether this is the case, we treated exponentially growing cells with 200 Wg ml31 nalidixic acid (Sigma-Aldrich) and examined cells 15 min later for GFP-L foci. Nalidixic acid is an inhibitor of DNA gyrase and topoisomerase IV, enzymes required for generating negative supercoils and unlinking DNA during replication.
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Fig. 4. Fluorescent micrographs of non-synchronised GFP-L expressing cells. DY329 L 6 s GFP-L cells were grown overnight in LB medium with 50 Wg ml31 kanamycin at 30‡C, diluted 200-fold in fresh LB, and grown to early exponential phase. At this point, cells were removed and examined by confocal microscopy. Representative images of cells containing one (A,B), two (C^F), three (G^J) and four (K,L) foci are shown. Scale bar, 2 Wm.
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cells can be studied. To obtain the data on the positions of the replisomes during the cell cycle of E. coli, we ¢rst examined the relative positions of GFP-L foci within cells undergoing exponential growth (OD600 = 0.6). Fig. 4 shows typical examples of £uorescence micrographs of GFP-L expressing cells grown in LB medium and the analysis of their intracellular locations is shown in Fig. 5. Shorter cells contained a single focus that was preferentially found near the cell midpoint (Figs. 4A,B, and 5A). Two foci were seen on either side of the midcell, often appeared to be symmetrically displaced from the centre and were often clustered at 1/4 and 3/4 positions (Figs. 4C^F and 5B). Three-focus cells predominantly had two foci located close together near the 1/4 position with the third focus at the 3/4 position (Figs. 4G^I and 5C). Cells containing four foci comprised only 4% of the population, and they usually appeared to have two pairs, where each pair was spaced similarly around the 1/4 and 3/4 positions (Figs. 4J^L and 5D). The number of foci per cell showed a strong correlation with cell length, which in E. coli is an approximate indicator of progression through the cell cycle [16]. The average lengths for cells containing one, two, three, and four foci were 1.98 U 0.34, 2.62 U 0.49, 3.43 U 0.57 and 3.73 U 0.58 Wm, respectively, which were all signi¢cantly di¡erent from each other (P 6 0.001; multiple range test). Despite the exponential growth in rich growth medium, many DY329 L 6 s GFP cells contained no visible foci and most cells had one or two foci (Fig. 3). The reason for this is not clear. Our data showed that the GFP-L expressing strain grew more slowly than the wild-type strain (DY329) having a 55-min doubling time compared to the 35-min doubling time of the wild-type when both strains were grown in LB at 30‡C (data not shown). These results suggest that cells without foci had non-replicating nucleoid and that multiple fork formation was rare. Alternatively we could not detect faint foci due to the high background of di¡used GFP-L £uorescence in the cytoplasm. To further examine the relationship between the cellular location of GFP-L and the time of DNA replication we Table 1 Analysis of GFP-L foci in cells synchronised for initiation of chromosome replication Time after 30‡C shifta (min)
Fig. 5. Localisation of GFP-L foci in non-synchronised cells. The same population as shown in Fig. 3 was used in the analysis but cells were ¢xed in 70% methanol before microscopy. Cell lengths and positions of £uorescent signals were measured and the results plotted. Each dot/triangle represents a GFP-L focus. Distribution patterns for A: one focus (88 cells); B: two foci (119 cells); C: three foci (52 cells); D: four foci (18 cells).
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c
0 (548) 10 (368) 25 (382) 40 (250) 55 (373)
Percentage of cells with n focib 0
1
2
3
4
99 28 14 53 100
1 65 5 13 61
^ 7 72 29 ^
^ ^ 61 2 ^
^ ^ 9 3 ^
a Cultures of PC2/GFP-L were subjected to three temperature shifts as described in the text. b The data are cumulative from two independent experiments, each of which gave similar results. c Total cells counted.
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Fig. 6. Intracellular localisation of GFP-L foci in cells synchronised for initiation of chromosome replication. PC2/GFP-L cells grown at 30‡C in LB plus thymine (50 Wg ml31 ) were shifted to the non-permissive temperature of 42‡C for 1 h to stop DNA replication initiation. To synchronously initiate DNA replication, the cells were shifted to 30‡C for 6 min before being transferred again to 42‡C to inhibit further initiation. A: The rate of DNA synthesis in GFP-L/PC2 cells at 42‡C after a short temperature shift. The average of three replicates is shown. B: Fluorescence images of cells progressing through the cell cycle at 42‡C after temperature shift. Times after the 30‡C temperature shift are indicated. Scale bar, 1 Wm.
analysed the number (Table 1) and location of GFP-L foci in cells synchronised for initiation of chromosome replication. To do this, we constructed GFP-L transductant derived from a temperature sensitive dnaC2 mutant (strain PC2). The DnaC protein forms a complex with DnaB (helicase) and helps load DnaB onto DNA. At the nonpermissive temperature (42‡C) the PC2 strain is inactive for replication initiation, but replication initiation can be restored upon transfer to 30‡C [11,12]. A culture of PC2/ GFP-L strain in exponential phase at 30‡C was transferred to 42‡C (to allow completion of current rounds of replication and synchronise cells), then transferred to 30‡C for 6 min (to allow a single round of replication to be initi-
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ated) before being shifted back to 42‡C. Before transfer to 30‡C, GFP-L was dispersed throughout the cells and there were no apparent foci in most cells (Fig. 6B). However, within 10 min after the temperature shift, rather fuzzy £uorescent GFP-L foci were observed in 72% of cells (Table 1) and these were usually recruited near midcell in short cells and near the quarter positions in longer, twofocus cells (Fig. 6B, 10 min, arrows). At 25 min, most (86%) cells had very bright, well-de¢ned foci of GFP-L. When there were two foci, these were located at cell quarter positions. Longer cells appeared to have two pairs of foci, where each pair was spaced similarly around the 1/4 and 3/4 positions (Fig. 6B). At 40 min, GFP-L foci were
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observed in 47% of cells and these foci had similar distribution pattern to those observed at 25 min, but the foci were less bright, more di¡use and in some cells the foci were starting to disappear (Fig. 6B, 40 min, arrow). At 55 min the £uorescence was dispersed throughout the cell (Fig. 6B) and rarely formed discrete foci. The peaks of DNA synthesis (Fig. 6A) are correlated with bright GFP-L clusters localised at cell quarter positions (Fig. 6B, 25 min). From these results we conclude that immediately following DNA replication initiation the replication machinery was ¢rst recruited near midcell position (or future midcell in longer cells). As DNA replication progresses the replication machinery splits and the submachines rapidly move apart to occupy the cell quarter positions and reside there for the duration of DNA replication. The fractions of cells with two foci at 10 min and those with four foci at 25 min (7% and 9% respectively, Table 1) are consistent with the interpretation that cells with two foci convert to cells with four foci. Because only one round of DNA replication occurred in these cells, the apparently moved foci cannot be re-initiation events. Rapid photobleaching of the GFP-L has made it impossible for us to carry out time-lapse studies to directly con¢rm that the GFP-L foci ¢rst form at the centre, separate and then move away. Our more direct detection of the replisome by use of GFP-L fusion o¡ers new insights into the behaviour of the replisomes in living cells. The replication machinery in E. coli does not remain in the centre of the cell until the terminus has been replicated as suggested by the stationary replisome model [7,8,17,18]. The experimental results are consistent with the translocating replication factories model [11^13]. Using indirect immuno£uorescence in ¢xed cells, it has recently been found that in the absence of DNA replication, L molecules were localised at or near the cell poles [11]. We did not observe the clustering of inactive L at cell poles, but rather the £uorescence was dispersed throughout the cells. This may re£ect the di¡erent methods used in the two studies. Alternatively, the overall high expression of GFP-L expressed from the strong lac promoter in our cells (Fig. 1C) may mask any aggregation of L molecules in the polar region. The mechanism that allows the replication machinery to migrate is not known, but the movement seems to be rapid (‘jumping’) in opposite directions to 1/4 and 3/4 positions during chromosome replication. This migration may be mediated by the chromosome segregation and/or by the inherent nature of cell division to generate a nucleoidfree zone at the cell centre for septum formation [19].
Acknowledgements We thank Drs Ross Tellam, Rob Moore and Professor John Mattick for critical reading of the manuscript and Professor Sota Hiraga for his suggestion to use E. coli PC2
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to synchronise for initiation of chromosome replication. This work was funded in association with a Corporate Fellowship to P.A.J. within a CSIRO Chief Executive Special Project, the Bioactive Molecules Initiative.
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