DNA damage induced in vivo by 7-methoxy-2-nitronaphtho[2,1-b]-furan (R7000) in the lacI gene of Escherichia coli

1998 ◽  
Vol 422 (2) ◽  
pp. 237-245 ◽  
Author(s):  
Philippe Quillardet ◽  
Didier Boscus ◽  
Eliette Touati ◽  
Maurice Hofnung
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Laci Gene

scholarly journals The Escherichia coli Mismatch Repair Protein MutL Recruits the Vsr and MutH Endonucleases in Response to DNA Damage

2009 ◽  
Vol 191 (12) ◽  
pp. 4041-4043 ◽  
Author(s):  
Yaroslava Y. Polosina ◽  
Justin Mui ◽  
Photini Pitsikas ◽  
Claire G. Cupples
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Mismatch Repair ◽  
Repair Protein ◽  
Mismatch Repair Protein

ABSTRACT The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.

scholarly journals Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells

2010 ◽  
Vol 107 (25) ◽  
pp. 11537-11542 ◽  
Author(s):  
G. Cuevas-Ramos ◽  
C. R. Petit ◽  
I. Marcq ◽  
M. Boury ◽  
E. Oswald ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Genomic Instability ◽  
Mammalian Cells

scholarly journals Intracellular Copper Does Not Catalyze the Formation of Oxidative DNA Damage in Escherichia coli

2006 ◽  
Vol 189 (5) ◽  
pp. 1616-1626 ◽  
Author(s):  
Lee Macomber ◽  
Christopher Rensing ◽  
James A. Imlay
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Hydroxyl Radical ◽  
Oxidative Dna Damage ◽  
Copper Toxicity ◽  
Radical Formation ◽  
Pcr Analysis ◽  
Intracellular Copper

ABSTRACT Because copper catalyzes the conversion of H2O2 to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H2O2 than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H2O2 damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H2O2 resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H2O2-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while E. coli responds to H2O2 stress by inducing iron sequestration proteins, H2O2-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism.

scholarly journals Replication Rapidly Recovers and Continues in the Presence of Hydroxyurea inEscherichia coli

2017 ◽  
Vol 200 (6) ◽  
Author(s):  
Samvel A. Nazaretyan ◽  
Neda Savic ◽  
Michael Sadek ◽  
Brandy J. Hackert ◽  
Justin Courcelle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Dna Replication ◽  
Ribonucleotide Reductase ◽  
Specific Inhibitor ◽  
Content Type ◽  
Ribonucleotide Reductases ◽  
The Stability ◽  
Deoxyribonucleoside Triphosphate

ABSTRACTIn both prokaryotes and eukaryotes, hydroxyurea is suggested to inhibit DNA replication by inactivating ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. In this study, we show that the inhibition of replication inEscherichia coliis transient even at concentrations of 0.1 M hydroxyurea and that replication rapidly recovers and continues in its presence. The recovery of replication does not require the alternative ribonucleotide reductases NrdEF and NrdDG or the translesion DNA polymerases II (Pol II), Pol IV, and Pol V. Ribonucleotides are incorporated at higher frequencies during replication in the presence of hydroxyurea. However, they do not contribute significantly to the observed synthesis or toxicity. Hydroxyurea toxicity was observed only under conditions where the stability of hydroxyurea was compromised and by-products known to damage DNA directly were allowed to accumulate. The results demonstrate that hydroxyurea is not a direct or specific inhibitor of DNA synthesisin vivoand that the transient inhibition observed is most likely due to a general depletion of iron cofactors from enzymes when 0.1 M hydroxyurea is initially applied. Finally, the results support previous studies suggesting that hydroxyurea toxicity is mediated primarily through direct DNA damage induced by the breakdown products of hydroxyurea, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.IMPORTANCEHydroxyurea is commonly suggested to function by inhibiting DNA replication through the inactivation of ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. Here, we show that hydroxyurea only transiently inhibits replication inEscherichia colibefore replication rapidly recovers and continues in the presence of the drug. The recovery of replication does not depend on alternative ribonucleotide reductases, translesion synthesis, or RecA. Further, we show that hydroxyurea toxicity is observed only in the presence of toxic intermediates that accumulate when hydroxyurea breaks down, damage DNA, and induce lethality. The results demonstrate that hydroxyurea toxicity is mediated indirectly by the formation of DNA damage, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.

scholarly journals Events associated with DNA replication disruption are not observed in hydrogen peroxide-treated Escherichia coli

2021 ◽  
Vol 11 (4) ◽  
Author(s):  
Chettar A Hoff ◽  
Sierra S Schmidt ◽  
Brandy J Hackert ◽  
Travis K Worley ◽  
Justin Courcelle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Replication Fork ◽  
Dna Lesions ◽  
Pyrimidine Dimers ◽  
Oxygen Sensitive ◽  
Recf Pathway ◽  
Similar Processing

Abstract UV irradiation induces pyrimidine dimers that block polymerases and disrupt the replisome. Restoring replication depends on the recF pathway proteins which process and maintain the replication fork DNA to allow the lesion to be repaired before replication resumes. Oxidative DNA lesions, such as those induced by hydrogen peroxide (H2O2), are often thought to require similar processing events, yet far less is known about how cells process oxidative damage during replication. Here we show that replication is not disrupted by H2O2-induced DNA damage in vivo. Following an initial inhibition, replication resumes in the absence of either lesion removal or RecF-processing. Restoring DNA synthesis depends on the presence of manganese in the medium, which we show is required for replication, but not repair to occur. The results demonstrate that replication is enzymatically inactivated, rather than physically disrupted by H2O2-induced DNA damage; indicate that inactivation is likely caused by oxidation of an iron-dependent replication or replication-associated protein that requires manganese to restore activity and synthesis; and address a long standing paradox as to why oxidative glycosylase mutants are defective in repair, yet not hypersensitive to H2O2. The oxygen-sensitive pausing may represent an adaptation that prevents replication from occurring under potentially lethal or mutagenic conditions.

scholarly journals Transposon-Mediated Linker Insertion Scanning Mutagenesis of the Escherichia coli McrA Endonuclease

2004 ◽  
Vol 186 (17) ◽  
pp. 5699-5707 ◽  
Author(s):  
Brian P. Anton ◽  
Elisabeth A. Raleigh
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Functional Organization ◽  
Development Program ◽  
Scanning System ◽  
Scanning Method ◽  
Terminal Domain ◽  
Modified Dna ◽  
K 12

ABSTRACT McrA is one of three functions that restrict modified foreign DNA in Escherichia coli K-12, affecting both methylated and hydroxymethylated substrates. We present here the first systematic analysis of the functional organization of McrA by using the GPS-LS insertion scanning system. We collected in-frame insertions of five amino acids at 46 independent locations and C-terminal truncations at 20 independent locations in the McrA protein. Each mutant was assayed for in vivo restriction of both methylated and hydroxymethylated bacteriophage (M.HpaII-modified λ and T4gt, respectively) and for induction of the E. coli SOS response in the presence of M.HpaII methylation, indicative of DNA damage. Our findings suggest the presence of an N-terminal DNA-binding domain and a C-terminal catalytic nuclease domain connected by a linker region largely tolerant of amino acid insertions. DNA damage inflicted by a functional C-terminal domain is required for restriction of phage T4gt. Disruption of the N-terminal domain abolishes restriction of both substrates. Surprisingly, truncation mutations that spare the N-terminal domain do not mediate DNA damage, as measured by SOS induction, but nevertheless partially restrict M.HpaII-modified λ in vivo. We suggest a common explanation for this “restriction without damage” and a similar observation seen in vivo with McrB, a component of another of the modified-DNA restriction functions. Briefly, we propose that unproductive site-specific binding of the protein to a vulnerable position in the λ genome disrupts the phage development program at an early stage. We also identified a single mutant, carrying an insertion in the N-terminal domain, which could fully restrict λ but did not restrict T4gt at all. This mutant may have a selective impairment in substrate recognition, distinguishing methylated from hydroxymethylated substrates. The study shows that the technically easy insertion scanning method can provide a rich source of functional information when coupled with effective phenotype tests.

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