Replication-Dependent Recruitment of the {beta}-Subunit of DNA Polymerase III from Cytosolic Spaces to Replication Forks in Escherichia coli

The ß-subunit of DNA polymerase III is located asone or two condensed clusters within the nucleoid-occupied spacein exponentially growing cells of Escherichia coli. When chromosomereplication is terminated after incubation at nonpermissivetemperature in a temperature-sensitive dnaC mutant, the ß-subunitis located in the cytosolic spaces of the cell poles.

INTRODUCTION

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Introduction

The dnaN gene in Escherichia coli encodes the ß-subunitof DNA polymerase III. A dimer of the ß-subunit formsthe DNA sliding clamp. The dimer is loaded to DNA in an ATP-dependentmanner catalyzed by the ${gamma}$ complex (a subassembly of DNA polymeraseIII) to form the preinitiation complex. In the next step, theDNA polymerase III core that catalyzes DNA synthesis associateswith the preinitiation complex to form the initiation complex.The ß-subunit exists as 300 to 5,000 dimers per cell(for reviews, see references 1, 6, 7, and 9). In this work,we have analyzed the subcellular localization of the ß-subunitin exponentially growing wild-type cells and temperature-sensitivednaC mutant cells synchronized for initiation of chromosomereplication.

Subcellular localization of the ß-subunit of DNA polymerase III and SeqA in exponentially growing cells.

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Subcellular localization of the...

We analyzed the subcellular localization of the ß-subunitof DNA polymerase III by indirect immunofluorescence microscopy(4) using rabbit anti-ß-subunit polyclonal antibody.In exponentially growing cells of strain YK1100 (a tryptophan-deficientmutant derived from W3110 [11]) at 37°C in M9 glucose mediumsupplemented with L-tryptophan (50 µg/ml), the ß-subunitformed one or two distinct condensed clusters within the nucleoid.Besides clear clusters, a portion of ß-subunit moleculeswas distributed in polar cytosolic spaces in some cells (Fig.1A and Table 1). The average length of cells with two ß-subunitclusters was longer than that of cells with one ß-subunitcluster (Table 1 and Fig. 2A). A portion (45.8%) of cells didnot have clear clusters of the ß-subunit, which wasdispersed throughout the whole cell. The average length (2.09± 0.58 µm [mean ± standard deviation]) ofcells without clear clusters was similar to that (1.98 ±0.62 µm) of total cells with one and two clusters (Table1 and Fig. 2A). This result eliminates the possibility thatthis type of cells without clusters was in a specific stageof the cell cycle, for example, the D period (the nonreplicationperiod under the slow-growth conditions at a 55- to 60-min doublingtime). Presumably, it is difficult to detect faint clusters,because of a high background of ß-subunit dispersedthroughout the whole cell.

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FIG. 1. Subcellular localization of the ß-subunit of DNA polymerase III and the SeqA protein in exponentially growing cells. Cells of strain YK1100 (11) growing in M9 medium supplemented with 0.5% glucose and 50 µg of L-tryptophan per ml at 37°C were fixed and analyzed by indirect immunofluorescence microscopy (4) with rabbit anti ß-subunit of DNA polymerase III polyclonal antibodies and rabbit anti-SeqA polyclonal antibodies. (A) Subcellular localization of the ß-subunit of DNA polymerase III (left). Pictures on the right show 4',6'-diamidino-2-phenylindole (DAPI)-stained cells. (B) Subcellular localization of the SeqA protein (left). Pictures on the right show DAPI-stained cells.

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TABLE 1. Statistical analysis of ß-subunit clusters in cells growing in different media

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FIG. 2. Histogram of cell length. Cells of YK1100 growing at 37°C in M9 medium supplemented with 0.5% of glucose and 50 µg of L-tryptophan per ml (A) or in L medium (B) were fixed and analyzed by immunofluorescence microscopy.

In a rich medium (L medium), cells had one, two, three, andfour clusters of the ß-subunit (Table 1). The doublingtime of the strain was approximately 30 min at 37°C in Lmedium, suggesting that multiforked replication occurred. Notethat the average length of the cells with three ß-subunitclusters was between the average length of cells with two clustersand that of cells with four clusters. This indicates that cellswith three clusters were in an intermediate stage between cellswith two clusters and cells with four clusters. A portion (27.8%)of cells did not have clear clusters of the ß-subunit,which was dispersed throughout the whole cell (Table 1). Theaverage length (3.06 ± 0.86 µm) of cells withoutclear clusters was similar to that (3.21 ± 0.85 µm)of total cells with one, two, three, and four clusters (Table1 and Fig. 2B). It is unlikely that this type of cells withoutclusters was in a specific stage of the cell cycle.

We previously reported that SeqA is localized as discrete fociin growing YK1100 cells. SeqA forms clusters with hemimethylatednascent DNA segments behind replication forks (4, 10; for areview, see reference 3). We therefore analyzed the subcellularlocalization of the SeqA protein as a landmark of hemimethylatednascent DNA segments in the cell cycle. As shown in Fig. 1B,one or two clear fluorescent foci of SeqA were observed in exponentiallygrowing cells in M9 glucose medium supplemented with L-tryptophan(50 µg/ml). A single SeqA focus was localized at the middleposition of the cell within a nucleoid. Two SeqA foci were localizedat the one-fourth and three-fourths positions of the cell. SeqAfoci were never localized at the cell poles or the septal site.In L medium, cells had one, two, three, and four SeqA foci.These results for SeqA foci were consistent with previous observations(4).

Comparison between the localization of ß-subunit of DNA polymerase III and that of SeqA protein in cells synchronized for initiation of chromosome replication.

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Comparison between the...

To examine whether the formation of condensed clusters of ß-subunitdepends on ongoing replication, we analyzed the localizationof clusters in cells synchronized for initiation of chromosomereplication. To synchronize the initiation of chromosome replication,we used a temperature-sensitive dnaC mutant strain, PC2 (2,5). The dnaC gene codes for the DnaC protein, which loads theDnaB helicase onto DNA (for a review, see reference 9). Thethermosensitive DnaC mutant protein of strain PC2 is inactiveat 42°C, but its activity is immediately recovered aftertransfer to 30°C (5). Cells of PC2 growing exponentiallyat the permissive temperature of 30°C in L medium supplementedwith thymine (50 µg/ml) were transferred to the nonpermissivetemperature of 42°C for 60 min to complete ongoing roundsof replication without initiation. After the incubation at 42°C,the ß-subunit was localized in cytosolic spaces atthe cell poles (Fig. 3A). Subsequently, the cells were incubatedat 30°C for 6 min to synchronously initiate chromosome replicationfrom the replication origin oriC, and then they were transferredagain to 42°C to inhibit further initiation of chromosomereplication. During incubation at 30°C for 6 min, chromosomereplication was initiated in more than 90% of the total cells(our unpublished data). Under these conditions, only one roundof chromosome replication is presumed to occur. After 6 minat 30°C, ß-subunit molecules were recruited fromthe polar cytosolic spaces to the nucleoid, resulting in a condensedcluster. A portion of ß-subunit molecules still remainedin the polar cytosolic spaces (Fig. 3A). At 15 min, ß-subunitmolecules were localized as two condensed clusters in the majorityof cells or as one cluster in the minority of cells within thenucleoid (Fig. 3A). At 42 min, ß-subunit moleculeswere either dispersed throughout the cells or localized againin the polar cytosolic spaces (Fig. 3A). DNA synthesis was analyzedas shown in Fig. 4. The incorporation rate of [³H]thymidineinto the acid-insoluble fraction increased immediately aftertransfer to 30°C for 6 min. The incorporation rate was almostnull after approximately 40 min as expected. We conclude thatß-subunit clusters are formed within nucleoid-occupiedspaces and that this mechanism depends on ongoing chromosomereplication. Molecules of the ß-subunit are certainlyrecruited at and near replication forks.

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FIG. 3. Comparison of subcellular localization between the ß-subunit of DNA polymerase III and the SeqA protein in cells synchronized for initiation of chromosome replication. To synchronize the initiation of chromosome replication, cells of the temperature-sensitive dnaC mutant strain (strain PC2) were exponentially grown in L medium supplemented with thymine (50 µg/ml) at 30°C and then transferred sequentially to 42, 30, and 42°C as shown in arrows. Cells were removed, fixed, and analyzed by indirect immunofluorescence microscopy. (A) Subcellular localization of the ß-subunit of DNA polymerase III (left). Pictures on the right show 4',6'-diamidino-2-phenylindole (DAPI)-stained cells. (B) Subcellular localization of the SeqA protein (left). Pictures on the right show DAPI-stained cells.

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FIG. 4. Rate of DNA synthesis in a synchronized culture. Strain KK488, which is a Thy⁺ transductant derived from PC2, was exponentially grown at 30°C in M9 medium containing 1% glucose, 0.5% Casamino Acids (Difco), and 100 µg of L-tryptophan (without thymine) per ml. The culture was synchronized for initiation of chromosome replication by triple temperature shifts as shown in Fig. 3. One milliliter of culture was removed and labeled with [methyl-³H]thymidine (3.1 TBq/mmol; 0.37 MBq/ml of culture) in the presence of deoxyadenosine (200 µg/ml of culture) for 10 min at 42°C. The labeling was stopped by the addition of 2 ml of ice-cooled 10% trichloroacetic acid (TCA). The acid-insoluble fraction was collected with a glass filter and washed six times with ice-cooled 5% TCA. Incorporation of [methyl-³H]thymidine into the acid-insoluble fraction was counted with a scintillation counter.

For the same synchronized culture of PC2, we also analyzed thesubcellular localization of SeqA. SeqA was distributed throughoutthe whole nucleoid after the 60-min incubation at 42°C (Fig.3B). During the incubation at 42°C, ongoing rounds of chromosomereplication are completed and the chromosomal DNA becomes fullymethylated. As a result, SeqA molecules, which bind preferentiallyto hemimethylated DNA, are distributed in the whole nucleoid.Six minutes after the temperature shift to 30°C, SeqA moleculeshad been recruited to a single focus located at the middle position.Fifteen minutes after the shift, the majority contained twoSeqA foci localized at the one-fourth and three-fourths positions(Fig. 3B), while a minority contained a single SeqA focus inthe midcell position. Forty-two minutes after the shift, mostcells had elongated and had a single SeqA focus in the middleof the nucleoid (Fig. 3B). The single SeqA focus presumablycorresponds to a cluster of SeqA-bound hemimethylated terminusDNA.

The present data suggest that in the absence of DNA replication,the unbound ß-subunit molecules are localized in thepolar cytosolic spaces. Following initiation of chromosome replication,a substantial fraction of ß-subunit molecules arerecruited into a single focus located in the middle of the nucleoid,sometimes with the rest of molecules in the polar cytosolicspaces. A translocating replication factories model was previouslyproposed (3, 5). In this model, paired replication apparatusesthat mediate the bidirectional chromosome replication are firstlocated close to each other at the middle position of the nucleoid.Subsequently, the replication apparatuses separate and migratein opposite directions during replication fork progression.Each replication apparatus migrates together with a SeqA-hemimethylatedDNA cluster that associates with the replication apparatus.The subcellular localization of ß-subunit moleculesshown in this work is consistent with this model. When temperature-sensitiveinitiation mutant cells are transferred back from nonpermissiveto permissive temperature, ß-subunit molecules mayfirst be recruited to the oriC region, which is localized inthe middle position of the cell (5), and then paired replicationapparatuses may be formed in the oriC region for bidirectionalreplication of the chromosome. These replication apparatusesare located close to each other in the middle of the nucleoidfor a substantial period. The closely linked replication apparatusesare observed as a single fluorescent cluster. Subsequently,the replication apparatuses separate and rapidly migrate inopposite directions up to the one-fourth and three-fourths cellularpositions together with SeqA-bound hemimethylated DNA clusters.The present results for localization of the ß-subunitof DNA polymerase III support the translocating replicationfactories model (5) in E. coli but not the fixed replicationfactories model (5, 8). In the latter, paired replication apparatusesof bidirectional replication would be localized at the midcellposition throughout the one round of chromosome replicationuntil termination, and new paired replication apparatuses ofthe second-round replication would be formed at the one-fourthand three-fourths positions (future division sites) and wouldexist at the same positions throughout the second-round replication.

ACKNOWLEDGMENTS

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We thank Chiyome Ichinose, Yuki Kawata, and Mizuho Yano forassistance in the laboratory.

This work was supported by grants from the Ministry of Education,Science, Sports, Culture and Technology of Japan, Japan Societyfor the Promotion of Science, and CREST.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kuhonji 4-24-1, Kumamoto 862-0976, Japan. Phone: 81-96-373-6578. Fax: 81-96-373-6582. E-mail: hiraga{at}gpo.kumamoto-u.ac.jp.

REFERENCES

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