The DinB•RecA complex of <i>Escherichia coli</i> mediates an efficient and high‐fidelity response to ubiquitous alkylation lesions

Cafarelli, Tiziana M.; Rands, Thomas J.; Godoy, Veronica G.

doi:10.1002/em.21826

Cited by 19 publications

(36 citation statements)

References 41 publications

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“…These additional regulators include RpoS, mentioned above (13); polyphosphate, which mediates a nutrient limitation response (25); GroE, which is part of the heat shock response (26); and HU, which modulates chromosome structure (27). Pol IV activity and processivity in vitro are enhanced by interacting with the beta clamp (28), and its mutagenic activity is also modulated by UmuD, RecA, and the transcription factor NusA (29)(30)(31).…”

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confidence: 99%

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

et al. 2015

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Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNAdamaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ϳ60% of the foci consisted of three Pol IV molecules, while ϳ40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ϳ70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

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Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

et al. 2015

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“…If this were the case, then 8-oxo-dG would not be required as a specific intermediate in MBR; any fork-stalling DNA-base damage traversed or extended from by translesion DNA polymerases would be expected to substitute for 8-oxo-dG. We tested whether other persistent DNA damage could bypass the requirement for ROS and 8-oxo-dG in MBR by using methyl methanesulfonate (MMS), which methylates DNA, forming mainly N 7 -methyldeoxyguanosine and N 3 -methyldeoxyadenosine [66], and ultraviolet-C light (UV-C), which creates pyrimidine dimers and 6–4 photoproducts (reviewed by [67]), all of which stall the replicative DNA Pol III and can be traversed by translesion DNA polymerases [68–70]. We pulse-treated starved cells with nonlethal doses of MMS or UV-C before plating on minimal lactose medium with 100mM TU to scavenge ROS.…”

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confidence: 99%

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli

et al. 2017

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Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators.

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“…As before (32, 41), we used "standing start" assays (13,32,41), where the first nucleotide added to the primer is the one opposite to the template lesion (see Fig. 5B for a diagram), to determine the in vitro activity of the DinB variants compared to native DinB on undamaged or 3-methyladenine (3-meA) analogue-containing templates.…”

Section: Resultsmentioning

confidence: 99%

Selection of dinB Alleles Suppressing Survival Loss upon dinB Overexpression in Escherichia coli

et al. 2014

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Escherichia coli strains overproducing DinB undergo survival loss; however, the mechanisms regulating this phenotype are poorly understood. Here we report a genetic selection revealing DinB residues essential to effect this loss-of-survival phenotype. The selection uses strains carrying both an antimutator allele of DNA polymerase III (Pol III) ␣-subunit (dnaE915) and either chromosomal or plasmid-borne dinB alleles. We hypothesized that dnaE915 cells would respond to DinB overproduction differently from dnaE ؉ cells because the dnaE915 allele is known to have an altered genetic interaction with dinB ؉ compared to its interaction with dnaE ؉ . Notably, we observe a loss-of-survival phenotype in dnaE915 strains with either a chromosomal catalytically inactive dinB(D103N) allele or a low-copy-number plasmid-borne dinB ؉ upon DNA damage treatment. Furthermore, we find that the loss-of-survival phenotype occurs independently of DNA damage treatment in a dnaE915 strain expressing the catalytically inactive dinB(D103N) allele from a low-copy-number plasmid. The selective pressure imposed resulted in suppressor mutations that eliminated growth defects. The dinB intragenic mutations examined were either base pair substitutions or those that we inferred to be loss of function (i.e., deletions and insertions). Further analyses of selected novel dinB alleles, generated by single-base-pair substitutions in the dnaE915 strain, indicated that these no longer effect loss of survival upon overproduction in dnaE ؉ strains. These mutations are mapped to specific areas of DinB; this permits us to gain insights into the mechanisms underlying the DinB-mediated overproduction loss-of-survival phenotype.A ll cells accumulate DNA damage that, if left unrepaired, will stall DNA replication due to the inability of high-fidelity DNA polymerases (Pol) to use lesion-containing DNA as templates, a potentially lethal event (1). To ensure survival, Escherichia coli responds to replication fork stalling by upregulating the expression of the SOS-regulated genes (1-3), among which are specialized low-fidelity DNA polymerase genes. These polymerases can perform translesion synthesis (TLS), consisting of both insertion opposite to and elongation from DNA lesions on the template strand (4). TLS can result in elevated mutagenesis. In fact, data suggest that mutations responsible for resistance to several classes of antibiotics require TLS DNA polymerases (5-8). Moreover, these DNA polymerases appear to play a role in the development of disorders such as cancer in metazoans (9-12).E. coli strains lacking the dinB gene (⌬dinB), encoding DinB (DNA Pol IV), one of the TLS DNA polymerases, are sensitive to nitrofurazone (NFZ) and 4-nitroquinoline-1-oxide (4-NQO) (13-15), reagents that generate persistent DNA lesions on the N 2 group of deoxyguanine (N 2 -dG), as well as alkylating agents such as methyl methanesulfonate (MMS) (14,16). Moreover, strains overproducing DinB undergo survival loss. It has been proposed that this is the result of lethal double-s...

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The DinB•RecA complex of Escherichia coli mediates an efficient and high‐fidelity response to ubiquitous alkylation lesions

Cited by 19 publications

References 41 publications

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli

Selection of dinB Alleles Suppressing Survival Loss upon dinB Overexpression in Escherichia coli

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