Prompt repair of hydrogen peroxide-induced DNA lesions prevents catastrophic chromosomal fragmentation

Abstract

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 ~2-fold, without affecting the survival. Once cyanide is removed, recombinational repair in E. coli can mend several double-strand breaks, but cannot mend ~100 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.

Potentiation of hydrogen peroxide toxicity: From catalase inhibition to stable DNA-iron complexes

“…A recent study of yet another strong H 2 O 2 potentiator, cyanide (25, 41), while further solidifying the critical role of iron, brings back into focus stable DNA-iron interactions and highlights yet another important aspect of this multifaceted toxicity — timely DNA repair.…”

“…1), soluble Fe(II) iron donates one electron to a hydrogen peroxide molecule, causing its decomposition: Fe(II) + H 2 O 2 ➔ Fe(III) + OH· + OH− that produces hydroxyl radical capable of reacting with any organic compound at diffusion rates (5, 22). Hydroxyl radicals kill via DNA damage, as indicated by the exquisite H 2 O 2 sensitivity of DNA repair mutants (23–25). Fenton’s reaction is the reason why otherwise relatively innocuous extracellular H 2 O 2 becomes a potent poison once inside the cell.…”

“…Instead, we found evidence for a specific multi-stage scenario (Fig. 2): 1) cyanide recruits (releases) iron from intracellular iron depots, ferritin being one such iron donor; 2) the recruited (released) iron is somehow delivered directly to DNA and forms stable iron-DNA complexes; 3) these DNA-bound iron atoms, if not promptly removed (for example, by the ferritin-like Dps proteins), catalyze ROS-generating Fenton’s reaction right on the most sensitive target of the cell, the chromosomal DNA; 4) cyanide further potentiates this DNA-self-targeted Fenton’s reaction in an unknown way; 5) expeditious repair of the resulting single-strand breaks and lesions (with which CN may also interfere) somehow facilitates iron removal from DNA, giving DNA one more chance to avoid imminent double-strand breaks, — otherwise catastrophic chromosomal fragmentation ensues (25, 41). Below we discuss some aspects of CN potentiation of H 2 O 2 toxicity.…”

“…Two binding sites for iron on DNA, one at the backbone, while the other at the bases, have been proposed (121–123). In fact, it was suggested on multiple occasions that when iron has a freedom to bind DNA in the cell, the bulk of lethal oxidative DNA lesions comes from such stable DNA-iron complexes (25, 124–128). Remarkably, the rate of formation of oxidative lesions at such stable DNA-iron complexes in cells, apparently limited by the very low intracellular concentration of H 2 O 2 , is slow enough to allow DNA repair to expedite iron removal from the damaged DNA, effectively eliminating formation of double-strand breaks.…”

“…Remarkably, the rate of formation of oxidative lesions at such stable DNA-iron complexes in cells, apparently limited by the very low intracellular concentration of H 2 O 2 , is slow enough to allow DNA repair to expedite iron removal from the damaged DNA, effectively eliminating formation of double-strand breaks. At the same time, when the concentration of H 2 O 2 increases during the treatment, it takes only a couple of minutes for such stable DNA-iron complexes to, figuratively speaking, “burn through” DNA, breaking both strands in the same location (25) (Fig. 2).…”