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

Abstract

The ␤-subunit of DNA polymerase III is located as one or two condensed clusters within the nucleoidoccupied space in exponentially growing cells of Escherichia coli. When chromosome replication is terminated after incubation at nonpermissive temperature in a temperature-sensitive dnaC mutant, the ␤-subunit is located in the cytosolic spaces of the cell poles.The dnaN gene in Escherichia coli encodes the ␤-subunit of DNA polymerase III. A dimer of the ␤-subunit forms the DNA sliding clamp. The dimer is loaded to DNA in an ATP-dependent manner catalyzed by the ␥ complex (a subassembly of DNA polymerase III) to form the preinitiation complex. In the next step, the DNA polymerase III core that catalyzes DNA synthesis associates with the preinitiation complex to form the initiation complex. The ␤-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 ␤-subunit in exponentially growing wild-type cells and temperature-sensitive dnaC mutant cells synchronized for initiation of chromosome replication.Subcellular localization of the ␤-subunit of DNA polymerase III and SeqA in exponentially growing cells. We analyzed the subcellular localization of the ␤-subunit of DNA polymerase III by indirect immunofluorescence microscopy (4) using rabbit anti-␤-subunit polyclonal antibody. In exponentially growing cells of strain YK1100 (a tryptophan-deficient mutant derived from W3110 [11]) at 37°C in M9 glucose medium supplemented with L-tryptophan (50 g/ml), the ␤-subunit formed one or two distinct condensed clusters within the nucleoid. Besides clear clusters, a portion of ␤-subunit molecules was distributed in polar cytosolic spaces in some cells (Fig. 1A and Table 1). The average length of cells with two ␤-subunit clusters was longer than that of cells with one ␤-subunit cluster (Table 1 and (Table 1 and Fig. 2A). This result eliminates the possibility that this type of cells without clusters was in a specific stage of the cell cycle, for example, the D period (the nonreplication period under the slow-growth conditions at a 55-to 60-min doubling time). Presumably, it is difficult to detect faint clusters, because of a high background of ␤-subunit dispersed throughout the whole cell.In a rich medium (L medium), cells had one, two, three, and four clusters of the ␤-subunit (Table 1) (Table 1). The average length (3.06 Ϯ 0.86 m) of cells without clear clusters was similar to that (3.21 Ϯ 0.85 m) of total cells with one, two, three, and four clusters (Table 1 and Fig. 2B). It is unlikely that this type of cells without clusters was in a specific stage of the cell cycle.We previously reported that SeqA is localized as discrete foci in growing YK1100 cells. SeqA forms clusters with hemimethylated nascent DNA segments behind replication forks (4, 10; for a review, see reference 3). We therefore analyzed the subcellular localization of the SeqA protein as a landmark of hemimethylated nascent DNA segments in the cell cycle. As shown in...

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