Overproduction of <i>Escherichia coli</i> DNA polymerase DinB (Pol IV) inhibits replication fork progression and is lethal

Uchida, Kaori; Furukohri, Asako; Shinozaki, Yutaka; Mori, Tetsuya; Ogawara, Daichi; Kanaya, Shigehiko; Nohmi, Takehiko; Maki, Hisaji; Akiyama, Masahiro

doi:10.1111/j.1365-2958.2008.06423.x

Cited by 71 publications

(151 citation statements)

References 66 publications

(96 reference statements)

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“…These contacts, however, do not seem to prevent Pol IV from accessing β; previous biochemical experiments with a fully reconstituted replisome have shown that Pol IV can replace Pol III (35). Furthermore, overexpression of Pol IV beyond SOS levels in cells has been shown to arrest replication and induce toxicity because of unregulated access of Pol IV to the replication fork (15,35,36). Removing the CBM residues alleviates Pol IV toxicity, whereas mutating the rim-contacting residues partially alleviates it (16).…”

Section: Discussionmentioning

confidence: 97%

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

Kath

Jergic

Heltzel

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Translesion synthesis (TLS) by Y-family DNA polymerases alleviates replication stalling at DNA damage. Ring-shaped processivity clamps play a critical but ill-defined role in mediating exchange between Y-family and replicative polymerases during TLS. By reconstituting TLS at the single-molecule level, we show that the Escherichia coli β clamp can simultaneously bind the replicative polymerase (Pol) III and the conserved Y-family Pol IV, enabling exchange of the two polymerases and rapid bypass of a Pol IV cognate lesion. Furthermore, we find that a secondary contact between Pol IV and β limits Pol IV synthesis under normal conditions but facilitates Pol III displacement from the primer terminus following Pol IV induction during the SOS DNA damage response. These results support a role for secondary polymerase clamp interactions in regulating exchange and establishing a polymerase hierarchy.single-molecule techniques | DNA replication | DNA repair | lesion bypass | DinB

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Section: Discussionmentioning

confidence: 97%

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

Kath

Jergic

Heltzel

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Since the first described cases of MMR saturation and error catastrophe (20,22), new insight has emerged into the physiology of stressed bacteria, including stress due to carbon source deprivation (37), antibiotic exposure (38)(39)(40), impaired replication fork progress (38,41), and dNTP pool depletion by the RNR inhibitor, hydroxyurea (42,43). Some of these stresses have been reported to result in the production of oxygen radicals or a mutator phenotype (38,39,42,44).…”

Section: Discussionmentioning

confidence: 99%

Hypermutability and error catastrophe due to defects in ribonucleotide reductase

Ahluwalia

Schaaper

2013

Proc. Natl. Acad. Sci. U.S.A.

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The enzyme ribonucleotide reductase (RNR) plays a critical role in the production of deoxynucleoside-5′-triphosphates (dNTPs), the building blocks for DNA synthesis and replication. The levels of the cellular dNTPs are tightly controlled, in large part through allosteric control of RNR. One important reason for controlling the dNTPs relates to their ability to affect the fidelity of DNA replication and, hence, the cellular mutation rate. We have previously isolated a set of mutants of Escherichia coli RNR that are characterized by altered dNTP pools and increased mutation rates (mutator mutants). Here, we show that one particular set of RNR mutants, carrying alterations at the enzyme's allosteric specificity site, is characterized by relatively modest dNTP pool deviations but exceptionally strong mutator phenotypes, when measured in a mutational forward assay (>1,000-fold increases). We provide evidence indicating that this high mutability is due to a saturation of the DNA mismatch repair system, leading to hypermutability and error catastrophe. The results indicate that, surprisingly, even modest deviations of the cellular dNTP pools, particularly when the pool deviations promote particular types of replication errors, can have dramatic consequences for mutation rates.T he enzyme ribonucleotide reductase (RNR) is a critical cellular factor for the synthesis and control of the deoxynucleoside-5′-triphosphates (dNTPs), the building blocks for DNA replication and DNA repair (1). Specifically, RNR is responsible for the reduction of the ribonucleotides to the corresponding deoxynucleotides. In many well-studied cell systems, like the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, or mammalian cells, this reduction takes place at the level of the nucleoside diphosphates (NDP → dNDP), and each of four separate NDPs (ADP, GDP, CDP, and UDP) can serve as RNR substrate (1, 2). Following reduction, the dNDPs are converted to the corresponding dNTPs by nucleoside diphosphate kinase (NDK) (3). The DNA precursor dTTP is not generated directly through this pathway; instead, it is produced from dCTP via dUTP (4).RNRs have been subdivided into several classes, depending on the type of radical used in the catalytic reaction (1, 5-7). The class Ia RNR, as present in E. coli, yeast, and mammalian cells, employs a tyrosyl radical. Structurally, these RNRs are tetramers composed of a dimer of a large subunit (R1) and a dimer of a small subunit (R2). The large subunits contain the catalytic site and two allosteric regulatory sites (termed specificity site and activity site), whereas the small subunit contains the essential tyrosyl radical. The activity site is located at the N terminus of the R1 subunit and functions as an "on-off" switch: ATP binding leads to an active enzyme, whereas dATP binding inhibits the enzyme. Hence, by monitoring the ATP/dATP ratio, the enzyme aims to ensure an overall dNTP level that is presumably optimal for DNA replication (1, 8).The R1 specificity site, is a binding site for dATP, ATP,...

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“…We have previously reported that replication fork progression was impeded when DinB was rapidly overproduced, upon arabinose addition, from the dinB gene under the control of the P BAD promoter (P BAD -dinB) on a multi-copy plasmid (Uchida et al, 2008). On the other hand, in E. coli cells that accumulate DinB more moderately from a single P BAD -dinB on the chromosome, DNA synthesis slowed gradually (Uchida et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

“…Beyond its established TLS function at replication forks, these large amounts of DinB have led to proposals for additional biological roles that include error-free processing of endogenously formed lesions such as alkylation (Bjedov et al, 2007) and glycation (Yuan et al, 2008), adaptive mutagenesis (McKenzie et al, 2001;Slechta et al, 2003;Tompkins et al, 2003), error-prone double-strand break repair (Ponder et al, 2005) and longterm survival during stationary phase (Yeiser et al, 2002). Recently, based on the suppression of replication fork progression by ectopic dinB overexpression, it has also been proposed that DinB plays a role as a molecular brake in eukaryotic checkpoint-like regulation of replication fork progression in E. coli (Uchida et al, 2008;Indiani et al, 2009;Langston et al, 2009). This fork-brake activity would provide extra time for a DNA lesion to be repaired before the fork encounters another lesion ahead.…”

Section: Introductionmentioning

confidence: 99%

<i>Escherichia coli</i> DinB inhibits replication fork progression without significantly inducing the SOS response

et al. 2012

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The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dosedependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.

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Overproduction of Escherichia coli DNA polymerase DinB (Pol IV) inhibits replication fork progression and is lethal

Cited by 71 publications

References 66 publications

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

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

Hypermutability and error catastrophe due to defects in ribonucleotide reductase

<i>Escherichia coli</i> DinB inhibits replication fork progression without significantly inducing the SOS response

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