Crystal Structure of the Catalytic α Subunit of E. coli Replicative DNA Polymerase III
Meindert H. Lamers, Roxana E. Georgescu, Sang-Gyu Lee, Mike O'Donnell and John Kuriyan
Abstract: Bacterial replicative DNA polymerases such as Polymerase III (Pol III) share no sequence similarity with other polymerases. The crystal structure, determined at 2.3 Å resolution, of a large fragment of Pol III (residues 1–917), reveals a unique chain fold with localized similarity in the catalytic domain to DNA polymerase β and related nucleotidyltransferases. The structure of Pol III is strikingly different from those of members of the canonical DNA polymerase families, which include eukaryotic replicative polymerases, suggesting that the DNA replication machinery in bacteria arose independently. A structural element near the active site in Pol III that is not present in nucleotidyltransferases but which resembles an element at the active sites of some canonical DNA polymerases suggests that, at a more distant level, all DNA polymerases may share a common ancestor. The structure also suggests a model for interaction of Pol III with the sliding clamp and DNA.
Figures: Art work
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Supplemental Data: Figure S1
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Figure S2b
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PDB Coordinates: Local copy: 2HNH, 2HQA; links in PDB: 2HNH, 2HQA
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Figure 1. Overview of DNA Pol III Structure

(A) Top view in stereo and (B) side view in stereo. The location of the active site residues in the Palm domain are indicated by black spheres and the location of the phosphate ion in the PHP domain by red spheres. (C) The core region of Pol III alone resembles a cupped righthand shape. (PHP domain and ring and little Finger subdomains removed.)

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Figure 2. The Pol III Active Site Is Similar to that of Pol β

Close up of the active sites of (A) E. coli Pol I (Beese et al., 1993); PDB code 1KLN, (B) E. coli Pol III, (C) human Pol β (Sawaya et al., 1997); PDB code 1BPY. The view is similar to that of Figure 1A. The catalytic aspartates are indicated as black sticks. A topology diagram for each Palm domain is given below. The other domains are indicated as F (Fingers), T (Thumb), P (PHP), and β2α motif. §Domain annotation for Pol β has been flipped to be consistent with that of canonical DNA polymerases.

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Figure 3. Pol III Palm Domain Cannot Bind DNA as Does Pol β and May Be Distantly Related to that of Pol I

(A) Top view of human Pol β bound to DNA (Sawaya et al., 1997); PDB code 1BPY. (B) Shown is the same view of Pol III with the DNA of Pol β placed into the structure. The modeled DNA runs into the helix of the β2α motif on top of the Palm domain (blue) and the stem of the Thumb domain (green). For clarity the PHP domain and ring and little Fingers subdomains have been removed. The star indicates the position of the active site in Pol β and Pol III. (C) Ribbon diagram of the Palm domain of Pol I (Beese et al., 1993), PDB code 1KLN, and (D) Pol III with the β2α motif is indicated in blue. A topology diagram of each is shown, with the two segments of the Palm domain colored differently. (E) The domain organizations of different DNA polymerases are shown. Domains with similar structures between polymerases families are colored in full colors. Domains with similar functions but different structures have the outline colored (i.e., Thumb and Fingers domain). Domains that are not related by function are colored in black and white. §Domain annotation for Pol β has been flipped to be consistent with the domain annotation of Pol I and Pol III. * indicates Bacterial Pol X. **Pol II has an N-terminal domain of unclear function. *** indicates “Polymerase Associated Domain,” also called “Wrist” or “little Finger domain.”

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Figure 4. Pol III PHP Active Site Is Similar to that of DHH Phosphoesterases

(A) Surface representation showing PHP, Thumb, and Palm domains (similar view as in Figure 1B). A narrow groove runs from the Palm domain into a shallow cavity in the PHP domain that has phosphate bound. (B) Detailed view of shallow cavity in PHP domain is shown. (C) Active site of Streptococcus mutans PPase II (Merckel et al., 2001) is shown. Green sphere indicates a Mg2+ ion and purple spheres Mn2+ ions. The overall structure of this protein is unrelated to that of the PHP domain of Pol III.

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Figure 5. Model for DNA Binding in Pol III

(A) Surface representation of Pol III, with DNA indicated, is shown. The view is similar to that in Figure 1A. (B) Close up of the DNA-exit path, looking down the DNA axis, is shown. Several positively charged residues (colored in yellow) form an arch that contacts the backbone of the DNA. Active site residues are colored in black. (C) Close up view of a surface representation of the active site with conserved residues are represented in green. The viewpoint is along the arrow in panel (A). (D) Same view of the active site is shown with the catalytic triad in black sticks and residues that may be involved in nucleotide binding in yellow.

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Figure 6. The C-terminal Region of Pol III, including the OB Domain, Is Required for β Stimulation

(A) Schematic diagram of the location of the β motifs in Pol III and the location of the deletions are shown. (B) Production of full-length M13 dsDNA after 20 s by core (Pol III, β clamp, exonuclease) complexes with different Pol III deletion constructs. (C) Isothermal titration calorimetry binding profile of clamp binding to full-length Pol III and deletion constructs. Time course radiograms are shown in Figure S4.

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Figure 7. Model of Pol III Binding to DNA, Sliding Clamp β, and Exonuclease

(A) Surface representation of polymerase and DNA, with C-terminal region of Pol III and sliding clamp β indicated schematically, is shown. (B) Interaction sites of exonuclease binding to Pol III, with binding region in Pol III (Wieczorek and McHenry, 2006) colored in yellow and position of exonuclease insertion in Pol C in red.

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Figure S1. SAXS reconstructions show evidence for a flexible domain in full length pol III.

(A) Three representative three dimensional shape reconstructions of full length pol III show a main body with a long flexible protrusion. (B) After truncation of the last 243 residues (pol III917), the reconstructions no longer show the long flexible region. This construct was subsequently used to solve the structure of pol III. (C) Comparison of experimental (red) and calculated (orange) X-ray scattering curves of Pol III917.

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Figure S2a.  Sequence alignment of 5 Pol III sequences from Gram- bacteria, 5 Pol III sequence from Gram+ bacteria and 5 Pol C sequences from Gram+ bacteria. Sequences were aligned with ClustalX (Thompson, 1997) and manually adjusted. Secondary structure elements of Pol III are indicated above the sequences as springs (α-helices) and arrows (β-strands). The domains of Pol III are indicated with colored lines over the sequences. Residues discussed in the text are labelled in bold. Sequences used are from: ecoli: Escherichia coli, salty: Salmonella typhimurium, vibch: Vibrio cholerae, haein: Haemophilus influenzae, theaq: Thermus aquaticus, deira: Deinococcus radiodurans, bacsu: Bacillus subtilis, staau: Staphylococcus aureus, mycle: Mycobacterium leprae, strpy: Streptococcus pyogenes, enfea: Enterococcus faecalis, lacsa: Lactobacillus salivarius.

Thompson, J. D., Gibson,T.J., Plewniak,F., Jeanmougin,F. and Higgins,D.G. (1997). The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24, 4876-4882.

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Figure S2b. 

ecoli: Escherichia coli, salty: Salmonella typhimurium, vibch: Vibrio cholerae, haein: Haemophilus influenzae, theaq: Thermus aquaticus, deira: Deinococcus radiodurans, bacsu: Bacillus subtilis, staau: Staphylococcus aureus, mycle: Mycobacterium leprae, strpy: Streptococcus pyogenes, enfea: Enterococcus faecalis, lacsa: Lactobacillus salivarius.

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Figure S2c. 

ecoli: Escherichia coli, salty: Salmonella typhimurium, vibch: Vibrio cholerae, haein: Haemophilus influenzae, theaq: Thermus aquaticus, deira: Deinococcus radiodurans, bacsu: Bacillus subtilis, staau: Staphylococcus aureus, mycle: Mycobacterium leprae, strpy: Streptococcus pyogenes, enfea: Enterococcus faecalis, lacsa: Lactobacillus salivarius.

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Figure S3. Surface representation of Pol III with modelled DNA.

Same view as in Figure 5A (A) Electrostatic potential with positive patch suited for nucleotide binding and positive arch that lines the exit path of the DNA indicated. Also indicated are residues in the fingers domain that are suited for DNA interaction. (B) Sequence conservation in Pol III. Conserved residues in 40 Pol III sequences are indicated in green.

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Figure S4. Production of full length M13 dsDNA by core (Pol III, β clamp, ε, and θ) using either full length Pol III or different deletion constructs of Pol III. Experimental setup is similar to that of Figure 6B, but reactions were quenched at different time points (5seconds to 20 minutes).

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Figure S5. 

Table S1. Data collection and phasing statistics

Table S2. Refinement statistics

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Art Work by Meindert Lamers