During chromosome synthesis in Escherichia coli, replication forks are blocked by Tus bound Ter sites on approach from one direction but not the other. To study the basis of this polarity, we measured the rates of dissociation of Tus from forked TerB oligonucleotides, such as would be produced by the replicative DnaB helicase at both the fork-blocking (nonpermissive) and permissive ends of the Ter site. Strand separation of a few nucleotides at the permissive end was sufficient to force rapid dissociation of Tus to allow fork progression. In contrast, strand separation extending to and including the strictly conserved G-C(6) base pair at the nonpermissive end led to formation of a stable locked complex. Lock formation specifically requires the cytosine residue, C(6). The crystal structure of the locked complex showed that C(6) moves 14 A from its normal position to bind in a cytosine-specific pocket on the surface of Tus.

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.

Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with [ 15 N]amino acids and suitable for analysis by NMR spectroscopy without chromatographic purification. A system based on an Escherichia coli cell extract was optimized with regard to protein yield and minimal usage of 15 N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to 1.8 mg of human cyclophilin A per mL of reaction medium were obtained by expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage k p R and p L promoters, when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen samples, each selectively labelled with a different 15 N-enriched amino acid, were produced and analysed directly by NMR spectroscopy after ultracentrifugation. Cross-peaks from metabolic by-products were evident in the 15 N-HSQC spectra of 13 of the samples. All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis. No significant transamination activity was observed except for [ 15 N]Asp, where an enzyme in the cell extract efficiently converted Asp fi Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition, the activity of peptide deformylase appeared to be generally reduced in the cell-free expression system.