Structure of a sliding clamp on DNA

Abstract

The structure of the E. coli beta clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the beta ring. The pronounced 22 degrees angle of DNA through beta may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.

Fig. 1. β Binds DNA Independent of the Circular Shape of the Clamp

(A) Either circular pET11a plasmid DNA (triangles) or linear DNA fragments (diamonds) produced by HaeIII digestion of the plasmid are titrated into a solution containing β^OG. (B) The experiment of panel A was repeated using the β monomer mutant. (C) Titration of an 18 bp duplex into β^OG. (D) Titration of a primed template (18/28) into β^OG. Curve fitting (A+BΠAB) yields the apparent K_d values shown in each plot.

Fig. 2. Co-crystal color screen and electron density of DNA in the β-DNA complex

(A) Co-crystals of E coli β with primed templates labeled at the 5′ terminus with either Cy5- (top row) or TAMN- (bottom row). (B) Electron density map of DNA in the interior of β; front and side-views. Fourier difference maps with (Fo - Fc) coefficients in red contoured at 1.6 σ. (C) Schematic of β-DNA interactions. Nucleotides are numbered in the standard way, starting with the 5′ end of the primer strand and continuing with the 5′ end of the template strand. The residues of β that interact with DNA (cut-off, 4.5 angstroms) are marked in red (protomer A) or blue (protomer B); residues in gray belong to the symmetry related β molecule. Color assignment of nucleotide bases are as follows: dT (blue), dA (red), dC (green) and dG (yellow). The gray icon surrounding residue 11 represents the binding pocket of the symmetry related β molecule. The underlined residues represent polypeptide backbone interactions with the DNA.

Fig. 3. Structure of the β-DNA Complex

A) Ribbon representation of the β-DNA complex, front and side-views. DNA is tilted ~22° from the C2 rotation axis of β. The Cy5 moiety is not shown for clarity, but is shown in Fig. S4. (B) Detailed view of R24 (top) and Q149 (bottom) in the β-DNA complex compared to the apo β structure (blue). (C) Replication assays using primed M13 ssDNA coated with SSB contain Pol III* and the indicated amount of either wt β (blue diamonds), β_Q149A (red squares), β_R24A (orange triangles), or β_R24A/Q149A (green circles). (D) Polymerase extension rate was determined using primed M13 ssDNA to which β, or mutant β, is first assembled on the DNA, followed by initiating synchronous chain extension. Reactions were quenched at the indicated times and products were analyzed in a native agarose gel. (E) The scheme illustrates the bead conjugated primed DNA in which SSB blocks ³²P-β from sliding off the end of the DNA. Clamp loading rate was assessed in assays using either β_WT (blue), β_Q149A (red), β_R24A (orange), or β_R24A,Q149A (green).

Fig. 4. Interaction of β with the ssDNA region of the primed site

(A) Crystal lattice showing two molecules of the β·DNA complex. The ssDNA makes a crystal contact between two molecules of β. (B) Surface representation of the charged residues that line the channel directing ssDNA to the hydrophobic protein binding site of β. Basic residues are colored blue. The protein binding site is shaded purple and subsites I and II (defined in (Burnouf et al., 2004) are indicated. (C) View of ssDNA (orange) positioned inside the hydrophobic protein binding pocket of β. Thy₁₁ and Thy₁₂ occupy sub-site I of the β hydrophobic pocket; the exposed R246, R240 side chains interact with the DNA phosphate backbone. Tyr153 and Tyr154 (green) stack with Ade15 and Thy₁₃, respectively.

Fig. 5. The ssDNA Binding Tyrosines of β a27e Important to Function

(A) Replication assays contain the indicated amount of Pol III* and β (or mutant β). The plot shows the quantitation of DNA synthesis using either: β_WT (blue diamonds), β_Y154A (red squares), β_Y153A (orange triangles), or β _Y154A/Y153A (green circles). (B) Polymerase extension rate was determined using primed M13 ssDNA to which β, or mutant β, is assembled on the DNA followed by initiating synchronous chain extension and native agarose gel analysis of products after quenching at the indicated times. (C) Bead based clamp loading rate assays were performed using either β_WT (blue), β _Y154A (red), β _Y153A (orange), or β _Y154A,/Y153A (green). For the purpose of comparison, the purple squares are the result using β_R24A,Q149A.

Fig. 6. Pol III C-terminal 9mer peptide competes with primed template ssDNA

The scheme illustrates the experimental design. Fluorescent β^AF555 is bound to primed DNA containing a quencher. A 9-mer Pol III α-C terminal peptide binds to the hydrophobic pocket of β. Therefore, if ssDNA occupies the same site, addition of the Pol III 9-mer peptide should displace the quencher-DNA and result in recovery of β^AF555 fluorescence. The result indicates that the 9-mer Pol III peptide results in recovery of β^AF555 fluorescence (green triangles), and thus competes with primed DNA for the hydrophobic pocket. Use of a blunt duplex with a quencher forms a small amount of β-DNA complex under these conditions, but is not affected by addition of 9-mer peptide (blue diamonds). This indicates that peptide competes with ssDNA, only present on the primed site.

Fig. 7. Possible functions of β-DNA contacts

(A) Proposed role of DNA binding during the clamp loading reaction. Left diagram: the clamp loader subunits form a spiral surface that binds an open clamp, twisting it into a lockwasher configuration. Primed DNA is attracted inside the circular clamp loader, thereby positioning DNA through the ring. Middle diagram: attractive contacts between β and DNA induces the clamp to close around DNA. The planar closed ring no longer matches the spiral shape of the clamp loader, thereby disconnecting β from clamp loader subunits. Right diagram: Template ssDNA binds the hydrophobic pocket of β, displacing δ subunit and completing the clamp loading reaction. (B) Proposed “placeholder” role for ssDNA-β contact. The contact between the template ssDNA and protein binding pocket of β may hold it near the 3′ terminus of DNA. Pol III connects to both protein binding sites on β. Thus, Pol III binding likely displaces ssDNA from the protein binding pocket of β, and this may release β from the ssDNA template for sliding on dsDNA. (C) Proposed role of DNA tilt in polymerase switching on β. Since β consists of two identical protomers, the DNA may alternate from one to the other, facilitating interaction with different DNA polymerases bound to the same clamp.