RESUMO
Thymineless death strikes cells unable to synthesize DNA precursor dTTP, with the nature of chromosomal damage still unclear. Thymine starvation stalls replication forks, whereas accumulating evidence indicates the replication origin is also affected. Using a novel DNA labeling technique, here we show that replication slowly continues in thymine-starved cells, but the newly synthesized DNA becomes fragmented and degraded. This degradation apparently releases enough thymine to sustain initiation of new replication bubbles from the chromosomal origin, which destabilizes the origin in a RecA-dependent manner. Marker frequency analysis with gene arrays 1) reveals destruction of the origin-centered chromosomal segment in RecA(+) cells; 2) confirms origin accumulation in the recA mutants; and 3) identifies the sites around the origin where destruction initiates in the recBCD mutants. We propose that thymineless cells convert persistent single-strand gaps behind replication forks into double-strand breaks, using the released thymine for new initiations, whereas subsequent disintegration of small replication bubbles causes replication origin destruction.
Assuntos
Replicação do DNA/genética , Proteínas de Escherichia coli/genética , Exodesoxirribonuclease V/genética , Recombinases Rec A/genética , Origem de Replicação/genética , Timina/metabolismo , Dano ao DNA , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonuclease V/metabolismo , Modelos Genéticos , Mutação , Recombinases Rec A/metabolismo , Origem de Replicação/efeitos dos fármacos , Timina/farmacologiaRESUMO
Starvation for DNA precursor dTTP, known as 'thymineless death' (TLD), kills bacterial and eukaryotic cells alike. Despite numerous investigations, toxic mechanisms behind TLD remain unknown, although wrong nucleotide incorporation with subsequent excision dominates the explanations. We show that kinetics of TLD in Escherichia coli is not affected by mutations in DNA repair, ruling out excision after massive misincorporation as the cause of TLD. We found that the rate of DNA synthesis in thymine-starved cells decreases exponentially, indicating replication fork stalling. Processing of stalled replication forks by recombinational repair is known to fragment the chromosome, and we detect significant chromosomal fragmentation during TLD. Moreover, we report that, out of major recombinational repair functions, only inactivation of recF and recO relieves TLD, identifying the poisoning mechanism. Inactivation of recJ and rep has slight effect, while the recA, recBC, ruvABC, recG and uvrD mutations all accelerate TLD, identifying the protection mechanisms. Our epistatic analysis argues for two distinct pathways protecting against TLD: RecABCD/Ruv repairs the double-strand breaks, whereas UvrD counteracts RecAFO-catalyzed toxic single-strand gap processing.
Assuntos
Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Timina/metabolismo , Cromossomos Bacterianos/genética , Fragmentação do DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Viabilidade Microbiana/genética , Modelos Genéticos , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutação , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Recombinação Genética , Resposta SOS em Genética/genéticaRESUMO
Duplex Sequencing (DS) is a next-generation sequencing methodology capable of detecting a single mutation among >1 × 10(7) wild-type nucleotides, thereby enabling the study of heterogeneous populations and very-low-frequency genetic alterations. DS can be applied to any double-stranded DNA sample, but it is ideal for small genomic regions of <1 Mb in size. The method relies on the ligation of sequencing adapters harboring random yet complementary double-stranded nucleotide sequences to the sample DNA of interest. Individually labeled strands are then PCR-amplified, creating sequence 'families' that share a common tag sequence derived from the two original complementary strands. Mutations are scored only if the variant is present in the PCR families arising from both of the two DNA strands. Here we provide a detailed protocol for efficient DS adapter synthesis, library preparation and target enrichment, as well as an overview of the data analysis workflow. The protocol typically takes 1-3 d.
Assuntos
Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Taxa de Mutação , DNA Mitocondrial , Biblioteca Gênica , Humanos , Reação em Cadeia da Polimerase/métodos , Fluxo de TrabalhoRESUMO
Recent evidence has implicated APOBEC3B as a source of mutations in cervical, bladder, lung, head and neck, and breast cancers. APOBEC enzymes normally function in innate immune responses, including those that target retroviruses, suggesting links between mutagenesis, immunity and viral infection in the process of cancer development.
Assuntos
Citidina Desaminase/genética , Mutagênese , Neoplasias/genética , Desaminase APOBEC-1 , Feminino , Humanos , Masculino , Antígenos de Histocompatibilidade MenorRESUMO
Hydroxyurea (HU) is a potent remedy against a variety of ailments and an efficient inhibitor of DNA synthesis, yet its pharmacology is unclear. HU acts in Escherichia coli by the same mechanism as it does in eukaryotes, via inhibition of ribonucleotide reductase. When examining a controversy about concentrations of HU that prevent thymineless death in E. coli, we found instability in HU solutions that avoided prior detection due to its peculiar nature. In contrast to freshly dissolved HU, which did not affect respiration and was bacteriostatic, 1-day-old HU solutions inhibited respiration and were immediately bactericidal. Respiration was inhibited by two gases, hydrogen cyanide (HCN) and nitric oxide (NO), whose appearance we detected in "aged" HU stocks by gas chromatography-mass spectrometry; however, neither gas was bactericidal. While determining the cause of toxicity, we found that HU damages DNA directly. We also demonstrated accumulation of peroxides in HU solutions by enzymatic assays, which explains the toxicity, as both NO and HCN are known to kill bacteria when combined with hydrogen peroxide. Remarkably, we found that bactericidal effects of NO+H(2)O(2) and HCN+H(2)O(2) mixtures were further synergistic. Accumulation of decomposition products in solutions of HU may explain the broad therapeutic effects of this drug.