- Research article
- Open Access
Crystal structure of a DNA polymerase sliding clamp from a Gram-positive bacterium
© Argiriadi et al; licensee BioMed Central Ltd. 2006
Received: 31 August 2005
Accepted: 10 January 2006
Published: 10 January 2006
Sliding DNA clamps are processivity factors that are required for efficient DNA replication. DNA polymerases maintain proximity to nucleic acid templates by interacting with sliding clamps that encircle DNA and thereby link the polymerase enzyme to the DNA substrate. Although the structures of sliding clamps from Gram-negative bacteria (E. coli), eukaryotes, archaea, and T4-like bacteriophages are well-known, the structure of a sliding clamp from Gram-positive bacteria has not been reported previously.
We have determined the crystal structure of the dimeric β subunit of the DNA polymerase III holoenzyme of Streptococcus pyogenes. The sliding clamp from this Gram-positive organism forms a ring-shaped dimeric assembly that is similar in overall structure to that of the sliding clamps from Gram-negative bacteria, bacteriophage T4, eukaryotes and archaea. The dimer has overall dimensions of ~90 Å × ~70 Å × ~25 Å with a central chamber that is large enough to accommodate duplex DNA. In comparison to the circular shape of other assemblies, the S. pyogenes clamp adopts a more elliptical structure.
The sequences of sliding clamps from S. pyogenes and E. coli are only 23% identical, making the generation of structural models for the S. pyogenes clamp difficult in the absence of direct experimental information. Our structure of the S. pyogenes β subunit completes the catalog of clamp structures from all the major sequence grouping of sliding clamps. The more elliptical rather than circular structure of the S. pyogenes clamp implies that the topological nature of encircling DNA, rather than a precise geometric shape, is the most conserved aspect for this family of proteins.
The bacterial DNA Polymerase III holoenzyme has the remarkable ability to polymerize long stretches of DNA at great speeds (~750 bases per second for the E. coli enzyme) without dissociating from its template [1–3]. The β subunit of the holoenzyme is required for efficient processivity in bacteria. This protein factor wraps around double-stranded DNA at primer-template junctions, where it serves as a sliding clamp to tether the polymerase enzyme to its DNA substrate (reviewed in [4–6]). A separate ATP-dependent protein complex called the clamp loader is required to place the sliding clamp on primer-template DNA (reviewed in [7–9]). Once loaded by this complex, the sliding clamp can confer processivity to the catalytic α subunit of the polymerase.
Structural analysis of the E. coli β subunit showed that the bacterial clamp is a head-to-tail dimer, with two protomers that form a closed ring . The diameter of the ring interior is large enough to accommodate duplex DNA. Biochemical studies indicate that one interface of the clamp is opened by the clamp loader to allow passage of a primed DNA into this interior . The bacterial clamp does not share readily detectable sequence similarity with the trimeric proliferating cellular nuclear antigen (PCNA) clamps of eukaryotes and archaea. Nevertheless, the overall ring-shaped architecture of sliding clamps is highly similar in bacterial, archaeal, and eukaryotic clamps [12–14] as well as those from bacteriophage [15, 16].
Results and discussion
The crystal structure of the β subunit from Streptococcus pyogenes was determined by multiple anomalous diffraction (MAD) at 2.6 Å resolution using datasets for three separate wavelengths close to the selenium absorption edge. A model built into the phased electron density maps was refined subsequently to 2.1 Å resolution against native data (See Materials & Methods). These crystals (space group P21) contain one β dimer in the asymmetric unit. A non-crystallographic two-fold relates two protomers to form the dimeric clamp. This internal symmetry facilitated the initial stages of model building and refinement. The two molecules are related by a 180° rotation about an axis perpendicular to the plane of the ring with a root mean square (r.m.s.) deviation of 0.46 Å for Cα atoms between the two protomers.
Consistent with the structure of the E. coli β clamp , the S. pyogenes clamp dimer is formed in a head-to-tail fashion. The sequence identity between the E. coli and S. pyogenes β clamp sequences is relatively low at 23% (Figure 1). Low sequence identity is a characteristic of sequence comparisons between bacterial clamps and trimeric clamps, such as those of T4-bacteriophage gp45 and PCNA [12–16]. Interestingly, the S. pyogenes β clamp dimer has a distorted oval shape, unlike the more circular shape of the E. coli β clamp. The distortion from a more circular shape is a feature of both crystallographically-independent subunits although we cannot absolutely rule out the influence of crystal packing on the shape of these clamps. The longer of the outer oval diameters is ~90 Å while the shortest diameter across the clamp is ~65 Å (Figure 2).
The overall organization of the S. pyogenes β subunit is very similar to that of the E. coli β subunit, with three distinct domains in each protomer. The secondary structure and topology of each of these domains is also very similar to each other; a pair of four-stranded antiparallel β sheets that bracket two antiparallel α helices make up the fundamental domain that is repeated three times per protomer (and six times per clamp). Domains II and III consist of approximately 110 residues. Domain I contains six more residues that are inserted in a loop region between strands β4 and β5. This region includes a small additional β-strand (β4b) not present in the E. coli structure (residues 66–70) and several residues that are disordered (residues 61–65) in both protomers of the S. pyogenes clamp. The additional β-strand is part of an antiparallel five-stranded β-sheet made up together of strands β1, β4b, β6, β7 and β8. We note that a region comparable to residues 66–70 of the S. pyogenes clamp forms a fifth β-strand in the structure of yeast PCNA .
When comparing the E. coli and S. pyogenes β clamp structures, there are differences in the relative positioning of domains and their respective interconnecting linkers that contribute to the elliptical shape of the S. pyogenes clamp. This results in an overall r.m.s. deviation for Cα atoms that is relatively large (2.0 Å) between the β clamp dimers from E. coli and S. pyogenes. The r.m.s.d. in Cα positions of the individual domains that make up the basic clamp fold is smaller (~1.4 Å; an overlay of domains II of the E. coli and S. pyogenes clamps is shown in Figure 2c).
The oval shape of the S. pyogenes clamp suggests that it is the topological property of surrounding the DNA template in order to maintain DNA-interactions with its cognate polymerase that is a fundamental and conserved aspect of bacterial β clamps. The subunit organization and secondary structural topology of the S. pyogenes clamp is very similar to that of the E. coli sliding clamp. Like in the E. coli clamp, the S. pyogenes subunits form a head-to tail dimmer, with a markedly positive electrostatic potential within the interior of the ring. The S. pyogenes clamp is therefore expected to readily accommodate duplex DNA within it.
Sample preparation and characterization
The Streptococcus pyogenes β subunit was overexpressed in BLR DE3 cells using a previously described expression plasmid (pETSpdnaN, ). Wild-type β subunit was grown in 12 liters LB broth at 37°C and selenomethionine-derivatized β was grown in 8 liters of minimal media supplemented with selenomethionine at 37°C. Both wild-type and selenomethionine cells were selected with the inclusion of ampicillin and induced with 1 mM IPTG at 15°C for 12–14 hours. The cells were resuspended, and lysed via French press in Buffer A (20 mM Tris pH 7.5, 0.1 mM EDTA, 10% glycerol, 5 mM DTT) and 1 M NaCl. In all subsequent purification steps, fresh 5 mM DTT was added to prevent oxidative degradation. The lysate was initially purified by an ammonium sulfate cut (0.3 grams of ammonium sulfate per mL of lysate). The pellet was then resuspended in Buffer A + 20 mM NaCl and dialyzed overnight against 2 L of the same buffer mixture. Protein was eluted through a Fast Flow Q column over a 10-column volume gradient from Buffer A + 50 mM NaCl to Buffer A + 500 mM NaCl. The pooled protein was diluted two-fold and eluted through an EAH-Sepharose 4B column over a 10-column volume gradient from Buffer A + 50 mM NaCl to Buffer A + 500 mM NaCl. S. pyogenes β fractions were pooled and precipitated again with ammonium sulfate to minimize the volume of eluted sample. The pellet was resuspended and dialyzed to adjust conductivity. An additional ion exchange step (Mono Q) removed minor impurities using a 10-column volume gradient from Buffer A + 50 mM NaCl to Buffer A + 500 mM NaCl. Size exclusion (Superdex 200) was a final purification step used only for the selenomethionine-derivatized protein. Wild-type and selenomethionine purified β subunit was concentrated to 20 mg/mL as determined by Bradford assay.
Crystallization and x-ray crystallography
Recombinant wild-type and selenomethionine Streptococcus β subunit were crystallized by equilibrating 1.0 μL of protein solution (20 mg/mL Streptococcus β subunit, Buffer A + 100 mM NaCl) with 1.0 μL of precipitant buffer (30% PEG 2000 MME, 100 mM sodium acetate pH 4.5, 200 mM ammonium sulfate) in a hanging drop suspended over a 1 mL reservoir of precipitant buffer at 20°C. Trapezoidal crystals with approximate dimensions 0.5 mm × 0.2 mm × 0.1 mm appeared within 5 days and diffracted X-rays to 2.1 Å resolution with synchrotron radiation. S. pyogenes β subunit crystallized in the spacegroup P21 having unit cell dimensions a = 79.1 Å, b = 74.7 Å, c = 82.8 Å, α = 90°, β = 118.6°, γ = 90° with one dimer in the asymmetric unit and 53.8% estimated solvent content.
Diffraction data were collected from 20% ethylene glycol flashed-cooled crystals by using synchrotron radiation at the Lawrence Berkeley National Laboratories, beamline 5.0.1. Data reduction was achieved using HKL2000 . Attempts to determine the structure by using the crystal structure of E. coli β subunit  by molecular replacement were unsuccessful due to low sequence identity. Experimental phases were determined using multiple anomalous diffraction (MAD) data collected at 2.5 Å resolution at three wavelengths close to the selenium absorption edge. Selenium atom positions were identified using the program Shake-and-Bake  and further refined with the program MLPHARE in the CCP4 suite  (8 selenium sites in total, 4 from each monomer). By using the structure of the E. coli β subunit  and the selenium-derived experimental phases, a phased translation function was calculated using CNS  to initially place a model into experimental density, calculated with solvent-flattened non-crystallographic symmetry averaged MAD phases at 3.0 Å resolution using the program DM . Domains I, II, and III of Streptococcus β were refined initially as rigid bodies at 3.0 Å. Subsequently, the model was rebuilt and refined against native 2.1 Å data using CNS  and O . Individual B-factors were refined and bulk solvent correction was applied, and 145 water molecules were built into the model. Because of disorder in the side-chains for residues Arg 254 and Thr 377 in one protomer, only main-chain atoms were included in the model. The final free and working R-values are 28.4% and 24.5% respectively. Coordinates have been deposited with the Protein Data Bank under the accession code 2AVT.
Contribution of authors
MAA carried out the expression, purification, crystallization, X-ray data collection and of the refinement of the S. pyogenes β clamp, and wrote the initial version of the manuscript. ERG finalized refinement of the structure, further developed the manuscript, and created the manuscript figures. IB cloned the protein expression construct and assisted with protein purification. MOD and JK conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
The authors thank David Jeruzalmi for the critical reading of this manuscript. This work was supported by grants from the NIH to J.K. (GM45547) and M.O.D. (GM38839).
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