Crystal structure of a DNA polymerase sliding clamp from a Gram-positive bacterium

Abstract

Background: 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.

Results: We have determined the crystal structure of the dimeric beta 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 approximately 90 A x approximately 70 A x approximately 25 A 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.

Conclusion: 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 beta 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.

Figure 1

Alignment of the sequences of the Streptococcus pyogenes and E. coli β subunits as done by BLAST . Secondary structure elements are labeled using the nomenclature first used for E. coli β in ref. [10]. An additional β-strand found only in domain I of the S. pyogenes clamp from residues 67–71 is labeled as β4b.

Figure 2

(A) Structure of the Streptococcus pyogenes β subunit. Ribbon representation of the S. pyogenes β subunit. (B) Comparison of S. pyogenes β subunit with E. coli β [10]. (C) Overlay of domain II of S. pyogenes (green) and E. coli (blue) β. Superposition was done with top3d [25], and figures were rendered with PyMol (DeLano Scientific).

Figure 3

(A) Model of Streptococcus pyogenes β clamp surrounding DNA. (B) Electrostatic surface of S. pyogenes β subunit. Two views of the clamp are shown that differ by a ~180° rotation about the vertical axis. Red indicates regions of negative electrostatic potential, white indicates neutral regions, and blue indicates positive regions. Figure rendered with GRASP [26, 27] and PyMol (DeLano Scientific). The position on the face of the S. pyogenes clamp where the δ subunit of the clamp loader would be expected to bind is indicated based upon the co-crystal structure of the E. coli proteins [18].

Figure 4

Differences in helix α1" geometry between in E. coli and S. pyogenes β. The dimeric interface of both β clamps distort of this helix (E. coli in blue, S. pyogenes in green). In the monomeric form of β (shown in yellow) crystallized bound to the δ subunit of the clamp loader [18], this helix is straight. The other protomer of S. pyogenes β is shown in grey for reference, with the sidechain of Phe81 shown in red. Figure rendered with with PyMol (DeLano Scientific).