Deep Analysis of Residue Constraints (DARC): identifying determinants of protein functional specificity.

Protein functional constraints are manifest as superfamily and functional-subgroup conserved residues, and as pairwise correlations. Deep Analysis of Residue Constraints (DARC) aids the visualization of these constraints, characterizes how they correlate with each other and with structure, and estimates statistical significance. This can identify determinants of protein functional specificity, as we illustrate for bacterial DNA clamp loader ATPases.

Electrostatic Interactions at the Dimer Interface Stabilize the E. coli β Sliding Clamp.

Sliding clamps are ring-shaped oligomeric proteins that encircle DNA and associate with DNA polymerases for processive DNA replication. The dimeric Escherichia coli β-clamp is closed in solution but must adopt an open conformation to be assembled onto DNA by a clamp loader. To determine what factors contribute to the stability of the dimer interfaces in the closed conformation and how clamp dynamics contribute to formation of the open conformation, we identified conditions that destabilized the dimer and measured the effects of these conditions on clamp dynamics.

Solution structure of an "open" E. coli Pol III clamp loader sliding clamp complex.

Sliding clamps are opened and loaded onto primer template junctions by clamp loaders, and once loaded on DNA, confer processivity to replicative polymerases. Previously determined crystal structures of eukaryotic and T4 clamp loader-clamp complexes have captured the sliding clamps in either closed or only partially open interface conformations. In these solution structure studies, we have captured for the first time the clamp loader-sliding clamp complex from Escherichia coli using size exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS).

Solution study of the Escherichia coli DNA polymerase III clamp loader reveals the location of the dynamic ψχ heterodimer.

Several X-ray crystal structures of the E. coli core clamp loader containing the five core (δ', δ, and three truncated γ) subunits have been determined, but they lack the ψ and χ subunits. We report the first solution structure of the complete seven-subunit clamp loader complex using small angle X-ray scattering.