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. 2012 Jul 15;523(2):135-43.
doi: 10.1016/j.abb.2012.04.013. Epub 2012 Apr 22.

Stabilization of the Escherichia Coli DNA Polymerase III ε Subunit by the θ Subunit Favors in Vivo Assembly of the Pol III Catalytic Core

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Stabilization of the Escherichia Coli DNA Polymerase III ε Subunit by the θ Subunit Favors in Vivo Assembly of the Pol III Catalytic Core

Emanuele Conte et al. Arch Biochem Biophys. .
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Abstract

Escherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: α (polymerase), ε (3'-5' exonuclease), and θ. Genetic experiments suggested that θ subunit stabilizes the intrinsically labile ε subunit and, furthermore, that θ might affect the cellular amounts of Pol III core and HE. Here, we provide biochemical evidence supporting this model by analyzing the amounts of the relevant proteins. First, we show that a ΔholE strain (lacking θ subunit) displays reduced amounts of free ε. We also demonstrate the existence of a dimer of ε, which may be involved in the stabilization of the protein. Second, θ, when overexpressed, dissociates the ε dimer and significantly increases the amount of Pol III core. The stability of ε also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of ΔdnaK strains leads to rapid depletion of ε, and (ii) overproduction of θ overcomes both the depletion of ε and the temperature sensitivity of the strain. Overall, our data suggest that ε is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the θ subunit through its prevention of ε degradation.

Figures

Fig. 1
Fig. 1
(A) Gel filtration chromatogram of soluble proteins extracted from E. coli BW25993 (empty circles) and from the same strain deleted for the holE gene (filled circles). For each sample, 5 mg of protein was loaded onto a Superdex 200 column (1.6 × 70 cm.). (B) Exonuclease activity of the fractions reported in panel A. Activity was assayed using 0.14 mL of each fraction and thymidine 5′-monophosphate p-nitrophenyl ester as substrate.
Fig. 2
Fig. 2
SDS–PAGE (Panel A) and bi-dimensional electrophoresis (Panel B) of an aliquot of fraction 40 as reported in Fig. 1A and B. Proteins featuring molecular mass from 20 to 35 kDa were extracted from gel slices (A) or spots (B), and precipitated with TCA. The pellets were washed with cold acetone, and finally resuspended in sample buffer. The arrows (Panel A) and the empty circles (Panel B) indicate the electrophoretic bands and spots from which proteins were identified by mass spectrometry (Tables 1 and 2, respectively). From the excised spots indicated in Panel B the following proteins were identified by mass-spectrometry: ε subunit (spot 1), mannose permease (spot 2), transaldolase (TalB, spot 3), and malate dehydrogenase (Mdh, spot 4). Panels C and D: western blots of fractions isolated by gel filtration of protein extracts isolated from E. coli TOP10/pBAD-ε213 (C) and TOP10/pBAD-ε186 (D) and, hence, containing overexpressed ε213 or ε186. Top numbers indicate the fractions analyzed, while the positions of molecular mass markers (25 and 19 kDa) are indicated with an arrow.
Fig. 3
Fig. 3
(A) Kinetics of growth of E. coli BW25993 ΔholE recA containing the empty pBAD vector (empty circles) or the pBAD-θ plasmid (filled circles) at 37 °C in LB medium. Arabinose was added to the culture medium 3.5 h after the inoculation. (B) Portions of gel filtration chromatograms (Superdex 200 column) obtained with soluble proteins extracted from E. coli BW25993 ΔholE recA and containing the empty pBAD vector or the pBAD-θ plasmid induced to overexpress θ for 1, 2, or 3 h (filled triangles, empty squares, filled circles, respectively). (C) Protein concentration (as determined using the micro-Bradford assay) of fractions isolated in the gel filtration experiments reported in Panel B. (D) Protein concentration of fractions of soluble proteins extracted from E. coli BW25993 ΔholE recA and containing the empty pBAD vector (empty circles) or the pBAD-θ plasmid (filled circles), and induced to overexpress θ for 3 h.
Fig. 4
Fig. 4
(A) E. coli BW25993 ΔholE recA containing the pBAD-θ plasmid was induced to overexpress θ for 1, 2, or 3 h. Proteins were extracted, subjected to gel filtration, and each fraction assayed for exonuclease activity. The fractions representing the maximum of each activity peak (cf. Fig. 1B), corresponding to DNA Pol-III holoenzyme, DNA Pol-III core, ε dimer, and free ε are presented. Black, gray, and dark-gray bars indicate samples induced to overexpress θ for 1, 2, and 3 h, respectively. Error bars represent standard deviation (n = 3). (B) Specific exonuclease activity detected in gel filtration fractions from protein extracts of E. coli BW 25993 ΔholE recA containing the empty pBAD vector (black bars) or the pBAD-θ (gray bars) plasmid. Induction of θ was maintained for 3 h. Error bars represent standard deviation (n = 3).
Fig. 5
Fig. 5
(A) Kinetics of growth of 25 mL cultures of E. coli ΔdnaK in LB low-salt medium. E. coli BW25993 ΔdnaK recA and containing the empty pBAD vector (empty circles), the pBAD-Hot (empty triangles), the pBAD-θ (filled triangles), or the pBAD-ε (empty squares) plasmid were grown for 5 h at 30 °C in arabinose-containing medium, then (see arrow) shifted to 42 °C. As a control, the growth kinetics of the dnaK+ isogenic strain is also reported (filled circles). (B) Kinetics of growth of 900 mL cultures of E. coli BW25993 ΔdnaK recA and containing the empty pBAD vector (empty circles) or the pBAD-θ construct (filled triangles); at the time indicated by the arrow, temperature was shifted to 42 °C and a sample (300 mL) of each culture was withdrawn. Double-headed arrows indicate the time at which further samples were harvested. Other conditions are as in A. Panels C and D: Specific exonuclease activity of fractions isolated from E. coli BW25993 ΔdnaK recA containing the empty pBAD or the pBAD-θ plasmid (black and white bars, respectively) and grown for 5 h at 30 °C in LB low-salt medium, and for a further 45 or 90 min at 42 °C (see Fig. 5B). Panels C and D respectively report the specific activity of the fractions representing the maximum of the activity peaks of dimeric and free ε. Error bars represent standard deviation (n = 3).

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