Insights Into Mutagenesis Using <i>Escherichia coli</i> Chromosomal <i>lacZ</i> Strains That Enable Detection of a Wide Spectrum of Mutational Events

Seier, Tracey; Padgett, Dana R.; Zilberberg, Gal; Sutera, Vincent A.; Toha, Noor Shamsiah; Lovett, Susan T.

doi:10.1534/genetics.111.127746

Cited by 33 publications

(67 citation statements)

References 63 publications

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“…Error bars represent 95% confidence intervals for mutation rates, mutations per cell generation. a white Lac − bacterial lawn (4). A blue halo of growth surrounding the discs impregnated with AZT provides a visual demonstration of the mutagenicity of the drug using the template-switch Lacreversion reporters, QP5 and QP6.…”

Section: Resultsmentioning

confidence: 99%

“…The mutational reporter strains used in this study have been described (4) and are derivatives of E. coli K-12 MG1655 and publicly available from the E. coli Genetic Stock Center (CGSC) (Yale University, New Haven, CT). The lexA3 mutation was introduced into mutational reporter strains constructed by P1 transduction with linked malF3190::Tn10-kan (strain STL6471), selecting kanamycin resistance and screening for UV sensitivity.…”

Section: Methodsmentioning

confidence: 99%

“…The mutations assayed include all base substitutions, frameshift mutations in nucleotide runs, deletions at microhomology and quasipalindrome-associated template-switch mutations (4). We assayed 12 or more strains and calculated mutation rates by the fluctuation methods described previously (4). Increases in mutation rate may be caused by two factors: increased likelihood of mutation incidence or decreased probability of its correction.…”

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confidence: 99%

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Azidothymidine and other chain terminators are mutagenic for template-switch-generated genetic mutations

Seier

Zilberberg²,

Zeiger³

et al. 2012

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

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The accumulation of mutations causes cell lethality and can lead to carcinogenesis. An important class of mutations, which are associated with mutational hotspots in many organisms, are those that arise by nascent strand misalignment and template-switching at the site of short repetitive sequences in DNA. Mutagens that strongly and specifically affect this class, which is mechanistically distinct from other mutations that arise from polymerase errors or by DNA template damage, are unknown. Using Escherichia coli and assays for specific mutational events, this study defines such a mutagen, 3′-azidothymidine [zidovudine (AZT)], used widely in the treatment and prevention of HIV/AIDS. At sublethal doses, AZT has no significant effect on frame shifts and most basesubstitution mutations. AT-to-CG transversions and deletions at microhomologies were enhanced modestly by AZT. AZT strongly stimulated the "template-switch" class of mutations that arise in imperfect inverted repeat sequences by DNA-strand misalignments during replication, presumably through its action as a chain terminator during DNA replication. Chain-terminating 2′-3′-didehydro 3′-deoxythymidine [stavudine (D4T)] and 2′-3′-dideoxyinosine [didanosine (ddI)] likewise stimulated template-switch mutagenesis. These agents define a specific class of mutagen that promotes template-switching and acts by stalling replication rather than by direct nucleotide base damage.] is a frontline drug in the treatment of HIV/AIDS. Its therapeutic effects arise by its incorporation during HIV reverse transcription, resulting in chain termination. Azidothymidine is genotoxic, particularly to mitochondria, presumably because mitochondrial DNA polymerase gamma readily incorporates AZT during DNA synthesis (reviewed in Ref. 1). However, AZT also induces formation of micronuclei, sister chromatid exchange events, and various chromosomal aberrations, suggesting it may also be incorporated into nuclear DNA at some level (2). Using the bacterium Escherichia coli as a model, we have shown that AZT blocks DNA replication and causes formation of single-strand DNA gaps and double-strand breaks (3). E. coli cells appear to tolerate a certain level of AZT by the combined action of the DNA damage response, homologous recombination, and exonuclease excision. The chain-terminating azidothymidine monophosphate does not appear to be removed by intrinsic DNA polymerase proofreading; rather, the exogenous 3′ to 5′ dsDNA exonuclease, exonuclease III (an ortholog of human APE1), is likely the enzyme that removes the residue from DNA during sublethal exposure, because ExoIII − (xthA) mutants are highly sensitive to the drug and have an enhanced DNA damage response (3). A 75 ng/mL dose of AZT reduces viability of xthA mutants about 10,000-fold; because this dose has negligible effects on wild-type cells, many AZT-monophosphate lesions can be removed by ExoIII to sustain replication and proliferative capacity.In this study we investigate the mutagenicity of AZT, at chronic, sublethal doses ∼100-fold ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Azidothymidine and other chain terminators are mutagenic for template-switch-generated genetic mutations

Seier

Zilberberg²,

Zeiger³

et al. 2012

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

Self Cite

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“…3). The design of additional probes specific for insertion and deletion mutations, 15 together with specific probes for mutational hotspots, such as quasipalindromes, 36 can further expand the use of this system for elucidating mutation and repair mechanisms.…”

Section: Discussionmentioning

confidence: 99%

A System for the Rapid Determination of the Mutation Spectrum in Escherichia coli

et al. 2012

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The first step toward elucidating the mutagenic effects of chemicals and pathways is to determine the specificity of the mutations generated spontaneously or in response to treatment with mutagens. We constructed a set of plasmid-encoded probes for the specific detection of each type of base substitution mutation. Using these probes, we were able to quickly determine both the mutation rate and the specificity of the mutations caused by different types of mutagens and mutagenic conditions. We also developed a PCR-based method to rapidly and robustly determine the mutation spectrum in response to various mutagenic samples in parallel. This system allows one to not only analyze the mutation specificity of various chemicals, but also to search for novel genetic elements that promote the specific mutation events.

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“…The appropriate gene deletion mutations were introduced into each of the six indicator strains by P1 transduction and checked by PCR with sitespecific primer pairs. We followed established protocols (27) to measure the frequency of Lac ϩ revertants for each type of point mutation. Mutation rates were calculated by using the MSS maximum-likelihood method (FALCOR package) (28,29).…”

mentioning

confidence: 99%

Genomewide Screen for Modulators of Evolvability under Toxic Antibiotic Exposure

Méhi

Bogos

Csörgő

et al. 2013

Antimicrob Agents Chemother

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Antibiotic resistance is generally selected within a window of concentrations high enough to inhibit wild-type growth but low enough for new resistant mutants to emerge. We studied de novo evolution of resistance to ciprofloxacin in an Escherichia coli knockout library. Five null mutations had little or no effect on intrinsic antibiotic susceptibility but increased the upper antibiotic dosage to which initially sensitive populations could adapt. These mutations affect mismatch repair, translation fidelity, and iron homeostasis. For many antimicrobial agents-including fluoroquinolones, cephalosporins, and rifamycins-a prominent factor contributing to the evolution of resistance is the acquisition of chromosomal mutations during therapy (1-3). While a wealth of highthroughput studies have investigated the impact of individual genes on intrinsic antibiotic susceptibility (4-6) or persistence (7), no systematic large-scale study has been devoted to the identification of molecular mechanisms that promote the evolution of antibiotic resistance.Here we systematically tested the impact of gene inactivation on the de novo evolution of antibiotic resistance. Escherichia coli is undoubtedly an ideal model prokaryote for such a study. The availability of a nearly complete single gene deletion library (the KEIO collection) enables the study of this issue in nearly all of the nonessential genes of this species (8). Our investigations concentrated on understanding the development of resistance to ciprofloxacin. It is one of the most widely deployed fluoroquinolone antibiotics in clinics, and its mechanism of action has been well studied (9-11).We developed a simple, high-throughput protocol that allows investigation of the de novo evolution of quinolone resistance in thousands of parallel bacterial cultures. Our goal was to identify genotypes (i.e., single-gene knockout strains) that permit adaptation to high antibiotic concentrations demanding the acquisition of one or more rare mutations (12, 13). All experiments were conducted with 96-deep-well plates containing 350 l LB medium supplemented with a toxic concentration of ciprofloxacin (200 ng/ml). The antibiotic dosage used is more than 10 times the MIC for wild-type (WT) E. coli (Table 1). About 10 8 cells were added to each well (two replicate populations per genotype). Following 5 days of incubation (37°C, 320 rpm), 2 l of each culture was transferred to an agar plate containing the same concentration of the antibiotic. After 24 h, the resistant bacteria of each genotype were counted. Initial positive hits (i.e., at least one resistant population per genotype) were validated with new sets of laboratory experiments. We used the same procedure as above with 96 replicate populations per genotype. Final hits were checked for the presence of the appropriate gene deletion by PCR using site-specific primers. At such a high ciprofloxacin concentration, the toxic effect of the antibiotic is substantial and population size rapidly declines (14). Typically, WT cultures became extinc...

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Insights Into Mutagenesis Using Escherichia coli Chromosomal lacZ Strains That Enable Detection of a Wide Spectrum of Mutational Events

Cited by 33 publications

References 63 publications

Azidothymidine and other chain terminators are mutagenic for template-switch-generated genetic mutations

Azidothymidine and other chain terminators are mutagenic for template-switch-generated genetic mutations

A System for the Rapid Determination of the Mutation Spectrum in Escherichia coli

Genomewide Screen for Modulators of Evolvability under Toxic Antibiotic Exposure

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