RESUMEN
Among strains of Escherichia coli that have evolved to survive extreme exposure to ionizing radiation, mutations in the recA gene are prominent and contribute substantially to the acquired phenotype. Changes at amino acid residue 276, D276A and D276N, occur repeatedly and in separate evolved populations. RecA D276A and RecA D276N exhibit unique adaptations to an environment that can require the repair of hundreds of double strand breaks. These two RecA protein variants (a) exhibit a faster rate of filament nucleation on DNA, as well as a slower extension under at least some conditions, leading potentially to a distribution of the protein among a higher number of shorter filaments, (b) promote DNA strand exchange more efficiently in the context of a shorter filament, and (c) are markedly less inhibited by ADP. These adaptations potentially allow RecA protein to address larger numbers of double strand DNA breaks in an environment where ADP concentrations are higher due to a compromised cellular metabolism.
Asunto(s)
Proteínas de Escherichia coli/genética , Mutación , Tolerancia a Radiación/genética , Rec A Recombinasas/genética , Reparación del ADN por Recombinación/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , ADN Bacteriano/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Radiación Ionizante , Rec A Recombinasas/antagonistas & inhibidores , Rec A Recombinasas/metabolismo , Reparación del ADN por Recombinación/fisiologíaRESUMEN
A transposon insertion screen implicated the yejH gene in the repair of ionizing radiation-induced damage. The yejH gene, which exhibits significant homology to the human transcription-coupled DNA repair gene XPB, is involved in the repair of double-strand DNA breaks. Deletion of yejH significantly sensitized cells to agents that cause double-strand breaks (ionizing radiation, UV radiation, ciprofloxacin). In addition, deletion of both yejH and radA hypersensitized the cells to ionizing radiation, UV and ciprofloxacin damage, indicating that these two genes have complementary repair functions. The ΔyejH ΔradA double deletion also showed a substantial decline in viability following an induced double-strand DNA break, of a magnitude comparable with the defect measured when the recA, recB, recG or priA genes are deleted. The ATPase activity and C-terminal zinc finger motif of yejH play an important role in its repair function, as targeted mutant alleles of yejH did not rescue sensitivity. We propose that yejH be renamed radD, reflecting its role in the DNA repair of radiation damage.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Reparación del ADN/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Rayos Ultravioleta , Adenosina Trifosfatasas/química , Alelos , Antibacterianos/farmacología , Ciprofloxacina/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Genes Bacterianos , Secuencias Invertidas Repetidas , Alineación de Secuencia , Eliminación de Secuencia , Dedos de ZincRESUMEN
To further an improved understanding of the mechanisms used by bacterial cells to survive extreme exposure to ionizing radiation (IR), we broadly screened nonessential Escherichia coli genes for those involved in IR resistance by using transposon-directed insertion sequencing (TraDIS). Forty-six genes were identified, most of which become essential upon heavy IR exposure. Most of these were subjected to direct validation. The results reinforced the notion that survival after high doses of ionizing radiation does not depend on a single mechanism or process, but instead is multifaceted. Many identified genes affect either DNA repair or the cellular response to oxidative damage. However, contributions by genes involved in cell wall structure/function, cell division, and intermediary metabolism were also evident. About half of the identified genes have not previously been associated with IR resistance or recovery from IR exposure, including eight genes of unknown function.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Radiación Ionizante , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Proteínas de Escherichia coli/genéticaRESUMEN
By directed evolution in the laboratory, we previously generated populations of Escherichia coli that exhibit a complex new phenotype, extreme resistance to ionizing radiation (IR). The molecular basis of this extremophile phenotype, involving strain isolates with a 3-4 order of magnitude increase in IR resistance at 3000 Gy, is now addressed. Of 69 mutations identified in one of our most highly adapted isolates, functional experiments demonstrate that the IR resistance phenotype is almost entirely accounted for by only three of these nucleotide changes, in the DNA metabolism genes recA, dnaB, and yfjK. Four additional genetic changes make small but measurable contributions. Whereas multiple contributions to IR resistance are evident in this study, our results highlight a particular adaptation mechanism not adequately considered in studies to date: Genetic innovations involving pre-existing DNA repair functions can play a predominant role in the acquisition of an IR resistance phenotype. DOI: http://dx.doi.org/10.7554/eLife.01322.001.
