Book of Abstracts: Albany 2005
Recognition of Primer-template DNA by a Clamp Loader Protein
The assembly of circular sliding clamps on primed templates is a critical feature of processive DNA replication. The clamp encircles DNA and binds the polymerase, tethering the enzyme to DNA to facilitate efficient synthesis. A clamp loader is responsible for binding the clamp, opening it, recognizing primer-template DNA and placing the clamp around it, where it can be utilized by the polymerase. Clamp loaders are multi-protein complexes that utilize ATP to fuel clamp assembly on DNA. It is not clear yet how these proteins select only primer-template DNA as the target for clamp assembly. To address this issue, we have analyzed the interaction between the E. coli clamp loader, γ complex, and DNA using UV-induced protein-DNA cross-linking and mass spectrometry. The results show that δ subunit in γ complex makes close contact with the primer-template junction, with Tryptophan-279 (W279) in the δ C-terminal domain located near the 3?-OH primer end. Previous studies have shown that δ also binds and opens the β clamp (hydrophobic residues in the N-terminal domain of δ contact β; Jeruzalmi et al., Cell 106, 417-428 (2001)). As shown in Figure 1, the clamp-binding and DNA-binding sites on d appear positioned for facile entry of the duplex portion of the primer-template into the clamp at the bottom of the complex and exit of the single-stranded template near the top of the complex. We consider it possible that W279 stacks against the final base pair at the primer-template junction and thus facilitates specific interaction between the clamp loader and primer-template (versus single-stranded or double-stranded DNA). In order to test this hypothesis, we have generated three W279 mutants, W279A, W279L, and W279Y, and are assaying them for DNA binding activity. Preliminary data indicate that the mutants with aliphatic amino acid substitutions (W279A and W279L) do not recognize primer-template DNA while the mutant with an aromatic amino acid substitution (W279Y) mimics the activity of the wild type protein, consistent with the hypothesis that a stacking interaction between the protein and DNA may play an important role in the selection of primed template DNA for clamp assembly.