DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a τ-Containing DnaX Complex to Enable Exchange into Replication Forks

Abstract

Examples of dynamic polymerase exchange have been previously characterized in model systems provided by coliphages T4 and T7. Using a dominant negative D403E polymerase (Pol) III ␣ that can form initiation complexes and sequester primer termini but not elongate, we investigated the possibility of exchange at the Escherichia coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a -containing DnaX complex did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of or the alternative shorter dnaX translation product ␥ showed that -, 2 -, or 3 -DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size, and gaps between fragments, possessed the ability to challenge pre-established replication forks, and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one . Previously, it was thought that at least two s in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed.As with all cellular replicases, the DNA polymerase (Pol) 2 III holoenzyme (HE) of Escherichia coli is tripartite with a sliding clamp processivity factor (␤ 2 ), a clamp loader (DnaX complex, DnaX 3 ␦␦'), and an associated replicative polymerase (Pol III, ␣⑀) (for a review, see Ref. 2). In E. coli and many other bacteria, DnaX encodes two products: a shorter ␥ protein that has ATPase activity and the ability to support clamp loading on single-stranded DNA and a longer protein that has additional domains that interact with the DnaB 6 replicative helicase and Pol III. Cells that cannot express ␥ exhibit defects in UV viability and Pol IV-mediated mutagenesis (3). A proposal has been made that ␥ may be required to avoid the presence of a third Pol III at the replication fork that might outcompete other polymerases, such as Pol IV, that must enter the replication fork to resolve unrepairable lesions and to perform stress-induced mutagenesis functions (2, 3).An E. coli rolling circle replication system has been developed that exploits a 409-nt flapped circular template that has a 50:1 asymmetric GC content, allowing convenient distinction, quantification, and labeling of leading and lagging strands. The asymmetric GC distribution allows specific slowing of lagging strand synthesis by substitution of dGDPNP for dGTP. The V max for insertion of this analog is lower than the natural nucleotide, and the K m is higher, allowing "dialing in" a desired elongation rate without reducing the nucleotide to a level where its concentration is depleted during the progress of the reaction. Because the lagging strand half of the Pol III HE cycles when a new primer is made, even when the pre...

A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome

“…Further work is necessary to deduce whether the proposed primase-to-polymerase switch via the DnaG-induced conformational change in the helicase represents a mechanism that is regularly utilized in OF synthesis, or whether it serves as a backup mechanism to handle roadblocks and obstructions, i.e., when priming on the leading strand becomes necessary or when there are delays in the recycling of the lagging-strand polymerase. The first possibility would appear to be in strongest agreement with the recent paradigm shift in the field proposing a rather stochastic behaviour of Pol III* at the replication fork, with new Pol III*s dynamically exchanging in the replisome while DnaB remains stably associated at the fork (Geertsema and van Oijen, 2013; van Oijen and Dixon, 2015;Yuan et al, 2016;Beattie et al, 2017;Lewis et al, 2017;Graham et al, 2017;Monachino et al, 2017). Interestingly, the T7-phage employs a similar strategy of accumulation of polymerases (gp5) on the helicase fused to the primase (gp4) for prompt primer handoff (Loparo et al, 2011;Geertsema et al, 2014).…”

“…Recent advances in the field have challenged the deterministic view of the replisome as a perfectly orchestrated machine, whereby a single Pol III*, stably bound to the replication fork, replicates DNA in a strictly ordered sequence of events (van Oijen and Dixon, 2015;Graham et al, 2017). Instead, frequent turnover of the Pol III* in the replisome has been observed (Yuan et al, 2016;Beattie et al, 2017;Lewis et al, 2017), suggesting that the helicase acts as the central organizing structure of the replisome as opposed to the polymerase. An explanation for this surprising level of plasticity can be found in the network of weak interactions that enable polymerases from solution to eventually replace those at the fork (Geertsema and van Oijen, 2013;Lewis et al, 2017).…”

Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

“…DnaE is displaced by PolC, and this must occur early in vivo , because DnaE is error-prone and efficient at lesion bypass ( 15 , 16 ). Previous studies showed that PolC early gains access to primers synthesized by the DnaG–DnaE complex on the lagging-strand ( 20 , 59 ). However, the exact mechanism of polymerase switch is unknown.…”

Single-molecule visualization of fast polymerase turnover in the bacterial replisome

“…Nevertheless, the introduction of high concentrations of catalytically dead Pol III* (still able to bind primed DNA) inhibits ongoing replication (Yuan et al, 2016). Reconciling these different observations, we here demonstrate the presence of a novel exchange mechanism that allows Pol III* to remain stably associated with the replisome under conditions of high dilution, yet facilitates rapid exchange at nanomolar concentrations.…”