We present single-molecule studies of the replication machinery of Escherichia coli and describe the visualization of individual E. coli DNA polymerase III (Pol III) holoenzymes engaging in primer extension and leading-strand synthesis. When coupled to the replicative helicase DnaB, Pol III mediates leading-strand synthesis with a processivity of 10.5 kb, 8-fold higher than that of primer extension by Pol III alone. Addition of the primase DnaG to the replisome causes a 3-fold reduction in the processivity of leading-strand synthesis, an effect dependent upon the DnaB-DnaG proteinprotein interaction rather than primase activity. A single-molecule analysis of the replication kinetics with varying DnaG concentrations indicates that a cooperative binding of 2-3 DnaG monomers to the propagating DnaB destabilizes the replisome. The modulation of DnaB helicase activity through the interaction with DnaG suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during slow primer synthesis on the lagging strand.Complete and accurate replication of DNA involves the coordinated activity of a large number of proteins. The replisome, the molecular machinery of DNA replication, unwinds the doublestranded DNA (dsDNA), synthesizes primers to initiate synthesis, and polymerizes nucleotides onto each of the two growing strands 1 . The replication system of Escherichia coli is ideal for studying the dynamic interplay among the various components at the replication fork. The enzymes of the E. coli replisome duplicate DNA with remarkable efficiency: the replication fork moves at a rate approaching 1000 nucleotides per second while maintaining coordination between continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand 1,2 . A fully functional replisome that displays all the fundamental enzymatic reactions characterizing DNA replication can be reconstituted in vitro with a limited number of purified key protein components: the DnaB helicase unwinds dsDNA; the DnaG primase synthesizes short oligoribonucleotides for priming of synthesis of the lagging strand; and the DNA polymerase III (Pol III) holoenzyme polymerizes nucleotides onto each nascent strand ( Fig. 1 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe Pol III holoenzyme is composed of three subassemblies: a core polymerase, sliding clamp, and clamp loader complex. The core polymerase is a heterotrimer of three subunits: α, the DNA polymerase; ε, proofreading exonuclease; and θ, which stabilizes ε 4 . The αεθ core is a poorly processive polymerase that only incorporates <20 nucleotides before dissociating from the primer-template 5 . However, when tethered to the sliding clamp, a ring-shaped homodimer of β subunits that encircles dsDNA, the processivity of the core increases dramatically to several kilobases (kb) at ~750 bp/s 5 . The loading of the β 2 clamp onto the primer/template strand requires opening of the ring by the γ multiprotein clamp-loading complex ...

During bacterial DNA replication, the DnaG primase interacts with the hexameric DnaB helicase to synthesize RNA primers for extension by DNA polymerase. In Escherichia coli, this occurs by transient interaction of primase with the helicase. Here we demonstrate directly by surface plasmon resonance that the C-terminal domain of primase is responsible for interaction with DnaB 6 . Determination of the 2.8-Å crystal structure of the C-terminal domain of primase revealed an asymmetric dimer. The monomers have an N-terminal helix bundle similar to the N-terminal domain of DnaB, followed by a long helix that connects to a C-terminal helix hairpin. The connecting helix is interrupted differently in the two monomers. Solution studies using NMR showed that an equilibrium exists between a monomeric species with an intact, extended but naked, connecting helix and a dimer in which this helix is interrupted in the same way as in one of the crystal conformers. The other conformer is not significantly populated in solution, and its presence in the crystal is due largely to crystal packing forces. It is proposed that the connecting helix contributes necessary structural flexibility in the primasehelicase complex at replication forks.

A cell‐free protein synthesis system from which the release factor RF1 has been selectively removed enables the facile incorporation of unnatural amino acids into proteins at difficult and multiple sites by optimized use of orthogonal tRNA/aminoacyl‐tRNA synthetase systems. 19F NMR spectroscopy of a protein labeled combinatorially with trifluoromethyl phenylalanine (red in picture) at multiple sites establishes resonance assignments with a minimal number of samples.

The C-terminal β-hairpin of NS2B (NS2Bc) in the dengue virus NS2B-NS3 protease is required for full enzymatic activity. In crystal structures without inhibitor and in the complex with bovine pancreatic trypsin inhibitor (BPTI), NS2Bc is displaced from the active site. In contrast, nuclear magnetic resonance (NMR) studies in solution only ever showed NS2Bc in the enzymatically active closed conformation. Here we demonstrate by pseudocontact shifts from a lanthanide tag that NS2Bc remains in the closed conformation also in the complex with BPTI. Therefore, the closed conformation is the best template for drug discovery.

Paramagnetic lanthanide ions present outstanding tools for structural biology by NMR spectroscopy. Here we show that the 3:1 complexes between dipicolinic acid and lanthanides are paramagnetic reagents which can site-specifically bind to a wide range of proteins without formation of a covalent bond. The observed pseudocontact shifts can be interpreted by a single magnetic susceptibility anisotropy tensor, enabling its use for structure refinements. The resonance assignment of the paramagnetic spectrum is greatly facilitated by the rapid exchange between bound and free protein, leading to gradual chemical shift changes as the protein is titrated with the paramagnetic dipicolinic acid complex. The association with the paramagnetic lanthanide leads to weak molecular alignment in a magnetic field so that the reagents can be used for the measurement of residual dipolar couplings without the need of protein modification or anisotropic alignment media. The protein samples can be recovered by simple dialysis.