Single-molecule studies of fork dynamics in Escherichia coli DNA replication

Abstract

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 ...

Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis

“…1 C and D) and rate ( Fig. 2A) of each synthesis step were generated from a large number of events; the data were in agreement with previous single-molecule experiments for Pol III (22) and bulk data for Pol IV (23). Pauses between synthesis steps were exponentially distributed, consistent with a single rate-limiting step, and we observed that increasing the concentration of Pol III from 5 to 30 nM reduced the pause length ( Fig.…”

“…S2 A-C, time constant τ decreases from 19.7 to 12.4 s). Biophysical and structural data suggest that only one Pol III binds the clamp dimer (4,18,24,25), arguing that pauses observed during synthesis result from stochastic dissociation of Pol III from the clamp and the diffusionlimited recruitment of a new polymerase from solution (22).…”

Single-Molecule Studies of the Replisome

“…Similar experiments were performed on the leading-strand synthesis complex of E. coli (104). Here also, the processivity of the leadingstrand synthesis complex (10.5 kb) was significantly higher than that of the DNA polymerase alone (1.4 kb), pointing to an increase in stability when more factors that provide additional protein-protein and DNA-protein interactions are added.…”

“…The frequency of interaction between DnaG and DnaB controls the length of the Okazaki fragments (116,124). Single-molecule experiments on the E. coli leading-strand synthesis complex demonstrated that the association between DnaG and DnaB resulted in a cessation of fork progression (104), analogous to the pausing behavior observed in the T7 system (64). The reduction in processivity was cooperative with respect to the primase concentration when primer synthesis was allowed (104).…”

Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression

“…Processivity estimates of the E. coli replisome from ensemble studies indicate a lower limit of 50 kb, and an average bulk rate of Ϸ500-700 nt/s at 37°C (6,7). Processivity measurements from other recent single-molecule studies differ over a wide range, from 3 kb to Ͼ80 kb (8,9). In each of these previous studies replication was performed in the presence of excess proteins that could replace a replisome protein that dissociates from DNA, and thus the true processivity of the replisome has not been examined.…”