RESUMO
Escherichia coli cells treated with a combination of cyanide (CN) and hydrogen peroxide (HP) succumb to catastrophic chromosome fragmentation (CCF), detectable in pulsed-field gels as >100 double-strand breaks per genome equivalent. Here we show that CN + HP-induced double-strand breaks are independent of replication and occur uniformly over the chromosome,-therefore we used CCF to probe the nucleoid structure by measuring DNA release from precipitated nucleoids. CCF releases surprisingly little chromosomal DNA from the nucleoid suggesting that: (i) the nucleoid is a single DNA-protein complex with only limited stretches of protein-free DNA and (ii) CN + HP-induced breaks happen within these unsecured DNA stretches, rather than at DNA attachments to the central scaffold. Mutants lacking individual nucleoid-associated proteins (NAPs) release more DNA during CCF, consistent with NAPs anchoring chromosome to the central scaffold (Dps also reduces the number of double-strand breaks directly). Finally, significantly more broken DNA is released once ATP production is restored, with about two-thirds of this ATP-dependent DNA release being due to transcription, suggesting that transcription complexes act as pulleys to move DNA loops. In addition to NAPs, recombinational repair of double-strand breaks also inhibits DNA release by CCF, contributing to a dynamic and complex nucleoid structure.
Assuntos
Cromossomos Bacterianos , Escherichia coli , Trifosfato de Adenosina/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Peróxido de Hidrogênio/metabolismoRESUMO
Hydrogen peroxide (HP) or cyanide (CN) are bacteriostatic at low-millimolar concentrations for growing Escherichia coli, whereas CN + HP mixture is strongly bactericidal. We show that this synergistic toxicity is associated with catastrophic chromosomal fragmentation. Since CN alone does not kill at any concentration, while HP alone kills at 20 mM, CN must potentiate HP poisoning. The CN + HP killing is blocked by iron chelators, suggesting Fenton's reaction. Indeed, we show that CN enhances plasmid DNA relaxation due to Fenton's reaction in vitro. However, mutants with elevated iron or HP pools are not acutely sensitive to HP-alone treatment, suggesting that, in addition, in vivoâ CN recruits iron from intracellular depots. We found that part of the CN-recruited iron pool is managed by ferritin and Dps: ferritin releases iron on cue from CN, while Dps sequesters it, quelling Fenton's reaction. We propose that disrupting intracellular iron trafficking is a common strategy employed by the immune system to kill microbes.
Assuntos
Cromossomos Bacterianos/genética , Cianetos/toxicidade , Fragmentação do DNA/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Ferro/metabolismo , Cromossomos Bacterianos/efeitos dos fármacos , Cromossomos Bacterianos/metabolismo , Sinergismo Farmacológico , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismoRESUMO
Hydrogen peroxide (H2O2) is unique among general toxins, because it is stable in abiotic environments at ambient temperature and neutral pH, yet rapidly kills any type of cells by producing highly-reactive hydroxyl radicals. This life-specific reactivity follows the distribution of soluble iron, Fe(II) (which combines with H2O2 to form the famous Fenton's reagent),Fe(II) is concentrated inside cells, but is virtually absent outside them. Because of the immediate danger of H2O2, all cells have powerful H2O2 scavengers, the equally famous catalases, which enable cells to survive thousand-fold higher concentrations of H2O2 and, in combination with adequate movement of H2O2 across membranes, make the killing H2O2 concentrations virtually impractical to generate in vivo. And yet, low concentrations of H2O2 are somehow used as an efficient biological weapon. Here we review several examples of how cells potentiate H2O2 toxicity with other chemicals. At first, these potentiators were thought to simply inhibit catalases, but recent findings with cyanide suggest that potentiators mostly promote the other side of Fenton's reaction, recruiting iron from cell depots into stable DNA-iron complexes that, in the presence of elevated H2O2, efficiently break duplex DNA, pulverizing the chromosome. This multifaceted potentiation of H2O2 toxicity results in robust and efficient killing.
Assuntos
Catalase/metabolismo , DNA/química , Peróxido de Hidrogênio/toxicidade , Ferro/química , Animais , Bactérias/efeitos dos fármacos , Catalase/antagonistas & inibidores , Humanos , Concentração de Íons de HidrogênioRESUMO
Iron-dependent oxidative DNA damage in vivo by hydrogen peroxide (H2O2, HP) induces copious single-strand(ss)-breaks and base modifications. HP also causes infrequent double-strand DNA breaks, whose relationship to the cell killing is unclear. Since hydrogen peroxide only fragments chromosomes in growing cells, these double-strand breaks were thought to represent replication forks collapsed at direct or excision ss-breaks and to be fully reparable. We have recently reported that hydrogen peroxide kills Escherichia coli by inducing catastrophic chromosome fragmentation, while cyanide (CN) potentiates both the killing and fragmentation. Remarkably, the extreme density of CN+HP-induced chromosomal double-strand breaks makes involvement of replication forks unlikely. Here we show that this massive fragmentation is further amplified by inactivation of ss-break repair or base-excision repair, suggesting that unrepaired primary DNA lesions are directly converted into double-strand breaks. Indeed, blocking DNA replication lowers CN+HP-induced fragmentation only â¼2-fold, without affecting the survival. Once cyanide is removed, recombinational repair in E. coli can mend several double-strand breaks, but cannot mend â¼100 breaks spread over the entire chromosome. Therefore, double-strand breaks induced by oxidative damage happen at the sites of unrepaired primary one-strand DNA lesions, are independent of replication and are highly lethal, supporting the model of clustered ss-breaks at the sites of stable DNA-iron complexes.
Assuntos
Cromossomos Bacterianos/genética , Fragmentação do DNA/efeitos dos fármacos , Reparo do DNA , Peróxido de Hidrogênio/farmacologia , Cromossomos Bacterianos/efeitos dos fármacos , Cianetos/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Desoxirribonuclease I/metabolismo , Interações Medicamentosas , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genéticaRESUMO
We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRCâ¼6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the "natural" CRC limit of â¼8 (cells divide more slowly); the "functional" CRC limit of â¼22 (cells divide extremely slowly); and the "tolerance" CRC limit of â¼64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter.