Evidence for Moonlighting Functions of the   Subunit of Escherichia coli DNA Polymerase III

Dietrich, Manuela; Pedró, Laura; García, Jesús; Pons, Miquel; Hüttener, Mário; Paytubi, Sònia; Madrid, Cristina; Juárez, Antonio

doi:10.1128/jb.01448-13

Cited by 7 publications

(3 citation statements)

References 54 publications

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“…Extensive genetic and biochemical studies, most of which performed in Escherichia coli as model organism or using the E. coli replicase in vitro, have elucidated the architecture and function of the three subcomplexes and the importance of their different subunits. The Pol III core is composed of the subunits α (the polymerase catalytic subunit), ε (the proofreading 3 -5 exonuclease), and θ, a nonessential subunit that appears to be specific to Enterobacteriaceae [4][5][6]. The ring-shaped β clamp (or sliding clamp), formed by a dimer of the β subunit, encircles the double-stranded DNA (dsDNA), slides along dsDNA, and tethers the Pol III core to the DNA, hence significantly increasing the processivity of DNA replication [7,8].…”

Section: Introductionmentioning

confidence: 99%

Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa

et al. 2022

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DNA polymerase III (Pol III) is the replicative enzyme in bacteria. It consists of three subcomplexes, the catalytic core, the β clamp, and the clamp loader. While this complex has been thoroughly characterized in the model organism Escherichia coli, much less is known about its functioning and/or its specific properties in other bacteria. Biochemical studies highlighted specific features in the clamp loader subunit ψ of Pseudomonas aeruginosa as compared to its E. coli counterpart, and transposon mutagenesis projects identified the ψ-encoding gene holD among the strictly essential core genes of P. aeruginosa. By generating a P. aeruginosa holD conditional mutant, here we demonstrate that, as previously observed for E. coli holD mutants, HolD-depleted P. aeruginosa cells show strongly decreased growth, induction of the SOS response, and emergence of suppressor mutants at high frequency. However, differently from what was observed in E. coli, the growth of P. aeruginosa cells lacking HolD cannot be rescued by the deletion of genes for specialized DNA polymerases. We also observed that the residual growth of HolD-depleted cells is strictly dependent on homologous recombination functions, suggesting that recombination-mediated rescue of stalled replication forks is crucial to support replication by a ψ-deficient Pol III enzyme in P. aeruginosa.

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Section: Introductionmentioning

confidence: 99%

Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa

et al. 2022

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“…Our results suggest that cnu expression utilizes different promoters for its growth‐dependent regulation, which could modulate the cellular level of Cnu during growth. As Cnu can be involved in other cellular processes such as transcription termination and biofilm formation, different utilization of the tandem cnu promoters could also play an essential part in dynamically adjusting the expression rate to meet cellular demand for Cnu.…”

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confidence: 99%

Tandem Promoters for Expression of Cnu, a Nucleoid Protein, in Escherichia coli

Lee

Kim

Choi

et al. 2019

Bulletin Korean Chem Soc

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In Escherichia coli, initiation of chromosomal replication occurs at the replication origin, oriC. The binding of various proteins to the replication origin regulates the initiation of chromosomal DNA synthesis. Previously, Cnu, which was also named YdgT, was identified as an oriC-binding protein. 1 Cnu shares extensive sequence similarity to the nucleoid-associated Hha/YmoA protein family. Cnu and Hha together form a complex with H-NS and the complex binds oriC. 1 Cnu interacts with H-NS and StpA (H-NS paralogue) and either Cnu-StpA or Hha-StpA interaction can protect StpA from Lon-mediated proteolysis. 2 As both the cnu and hha mutation lead to the reduction of origin content, Cnu is supposed to participate in the regulation of the replication initiation. 1 Considering that replication initiation is coupled to growth rate, cnu expression should be regulated in accordance to growth stages. However, it has not known yet how cnu expression is regulated. In an effort to address this question, we examined promoter regions responsible for cnu transcription. For this purpose, we used primer extension analysis to determine 5 0 ends of cnu mRNA. Total RNAs prepared from exponentially growing or stationary-phase E. coli cells were used for primer extension analysis ( Figure 1). We found six major primer extension products whose 5 0 ends correspond to −59/−60, −96, −118/−119, −136, −143, and − 184 relative to the ATG start codon of Cnu. A homology search of the upstream sequences disclosed three promoter regions P1, P2, and P3, whose predicted transcription start sites well match to −59/−60, −136, and − 184, respectively. Since the other primer extension products to positions −96, −118/−119, and − 143 were not at the predicted transcription start sites, they might be produced from degradation products of the upstream transcripts. Therefore, the primer extension analysis suggests that cnu transcription occurs at the three tandem promoters. P3 transcription was prominent in the exponential phase, while P1 was active in the stationary phase. P2 transcription is functional in both the exponential and stationary phases.We then examined further in detail how cnu expression is regulated during growth. Total RNAs were prepared from cell cultures at different growth stages and subjected to primer extension analysis ( Figure 2). P3 was highly active in the mid-exponential phase (5 h growth), but its (a) (b) (c) Figure 1. Determination of 5 0 ends of cnu mRNA. (a) Identification of primer extension products. JM109 cells containing plasmid pHL355-36 were used for the preparation of total cellular RNAs. The 32 P-labeled primer Ext was used for primer extension. The products were analyzed by 8% denaturing polyacrylamide gel electrophoresis. The sequencing ladders (G, A, T, and C) were prepared with primer Ext. The numbers on the left side of the gel indicate the 5 0 end positions. E and S stand for exponentially growing and stationary-phase cells, respectively. (B) Schematic map of the cnu gene and its tandem promoters. (C) Sequences o...

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“…crescentus também é um organismo extremamente interessante quando se trata de genética. A holoenzima DNA Polimerase III ainda não foi caracterizada nesta bactéria, porém, análises comparativas entre os genomas sequenciados e depositados em bancos de dados até o momento mostram que a subunidade θ de E. coli, codificada pelo gene holE, que tem sua função associada com a estabilização da subunidade ε (dnaQ) no cerne catalítico e ligação dos componentes da replicase (Taft-Benz & Schaaper, 2004), está confinada somente no grupo das enterobacterias (Dietrich et al, 2014). Logo, estudos usando como modelo outra Pol III são limitados para o entendimento do funcionamento desta holoenzima para a síntese de DNA livre e propensa a erro em procariotos bacterianos (Jensen et al, 2001).…”

Section: -Algumas Bactérias Utilizam Outra Pol III Para Fazer Tls: O unclassified

Estudo da síntese translesão em <i>Caulobacter crescentus</i>.

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As the integrity of information contained in DNA is of utmost importance, it receives protection against harmful agents that may harm its structure. Even in case of damage, the cell has a group of proteins that are involved in the correction and mitigation of these damages. The first group is a set of proteins involved in error-free DNA repair. If these proteins fail to minimize damage, another set of proteins is expressed as an alternative to repair. Among these are DNA polymerases that specialize in using a damaged DNA strand as a template for replication. This mechanism enables the cell to survive potentially cytotoxic damage at the expense of mutagenesis. In bacteria, the DNA damage response involves a set of proteins that are expressed as part of the SOS response. Among them are enzymes involved in translesion synthesis (TLS). Unlike Escherichia coli that has three TLS error-prone polymerases, Caulobacter crescentus bears the imuABC mutagenic cassette that is involved in DNA synthesis using a damaged template. In this work, we studied the mechanism of TLS mediated by ImuABC in this bacterium, and we found a number of differences relative to the characteristics of the principal polymerase involved in TLS in E. coli (Pol V). ImuABC proteins when expressed at maximum levels of the SOS response are not able to increase the rates of spontaneous mutagenesis. ImuABC, unlike Pol V, does not require RecA to perform TLS. The presence of these proteins in a background without the recA gene is sufficient for UVC-induced mutagenesis to occur. In studying mutagenesis as a response to DNA damage induced by UVC radiation at genomic levels in C. crescentus, we noted that most of the mutations found are present in regions that have adjacent pyrimidines, which are known to be extremely reactive to UVC radiation, leading to the formation of photoproducts. Our data suggest that there is a region on the circular chromosome of C. crescentus that is preferably mutated, and this accumulation of mutations may be a consequence of the repair occurring near the replicative origin, leaving the accumulated mutations close to the replication termination region.

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Evidence for Moonlighting Functions of the Subunit of Escherichia coli DNA Polymerase III

Cited by 7 publications

References 54 publications

Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa

Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa

Tandem Promoters for Expression of Cnu, a Nucleoid Protein, in Escherichia coli

Estudo da síntese translesão em <i>Caulobacter crescentus</i>.

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