Nitric oxide (NO.
) is a versatile molecule that is critical for numerous biological processes. NO.
is created intracellularly by nitric oxide synthetases through oxidation of the guanido group of arginine. At low concentrations, NO.
mediates intracellular signaling and neurotransmission, while at higher concentrations, NO.
is used by activated macrophages to fight invading pathogens and tumor cells (25
). Although activated macrophages excrete a variety of agents, including reactive oxygen species, NO.
is considered to be a critical agent, because inhibition of nitric oxide synthetases strongly diminishes the cytotoxic effect of macrophages (19
and other reactive oxygen species are potent DNA-damaging agents, and exposure to NO.
has been shown previously to be cytotoxic and mutagenic in Escherichia coli
and a variety of other cell types (6
). Furthermore, NO.
can induce homologous recombination in E. coli
). While the mutagenic properties of NO.
have been studied extensively (14
), little is known about the underlying mechanism by which NO.
-induced DNA damage leads to homologous recombination and puts cells at risk of genetic rearrangements.
itself is not very reactive with DNA. However, NO.
can react with oxygen and superoxide to create N2
and peroxynitrite, potent DNA-damaging agents. N2
can deaminate DNA bases to create mutagenic lesions, such as uracil, hypoxanthine, and xanthine. Peroxynitrite is an oxidizing agent, which reacts preferably with guanine. This reaction primarily results in 8-oxoguanine, which is potentially mutagenic, and 8-nitroguanine, which is susceptible to spontaneous depurination (22
). Interestingly, 8-oxoguanine is more susceptible to oxidation by peroxynitrite than is guanine, resulting in secondary oxidation products, which are potentially mutagenic and cytotoxic (5
). Peroxynitrite has also been shown previously to induce single-strand breaks in plasmid DNA in vitro, most likely through the oxidative breakdown of deoxyribose (15
The base excision repair (BER) pathway plays a major role in the removal of bases with NO.
-induced damage in E. coli
). DNA glycosylases initiate BER by cleaving the N-glycosylic bond between the base and the deoxyribose, resulting in an abasic (AP) site. Subsequently, AP endonucleases incise the DNA backbone immediately 5′ to the AP site, to create a 3′-OH terminus and a 5′-deoxyribose phosphate residue. The 5′-deoxyribose phosphate residue is removed by a deoxyribose phosphodiesterase, while the 3′-OH is extended by DNA polymerase I. Repair is then completed by a DNA ligase (31
). DNA glycosylases that can remove oxidative base damage have an associated lyase activity, so that removal of the base is thought to be coupled with nicking of the DNA backbone on the 3′ side of the lesion, resulting in a cleaved AP site. While this process eliminates the need for a deoxyribose phosphodiesterase, an AP endonuclease activity is still required to generate the 3′-OH terminus, necessary for DNA synthesis.
Several DNA glycosylases are potentially involved in the removal of NO.
-induced lesions in E. coli
. Uracil, xanthine, hypoxanthine, and 8-oxoguanine are substrates of the uracil DNA glycosylase (Ung), endonuclease V DNA glycosylase, AlkA 3-methyladenine DNA glycosylase (AlkA), and formamidopyrimidine DNA glycosylase (Fpg or MutM), respectively (18
). While Ung has been shown previously to protect against NO.
-induced mutations, no DNA glycosylase-deficient strains, including the ung
mutant, have been found to have enhanced sensitivity to NO.
). In contrast, E. coli
strains deficient in AP endonuclease activity (double mutant in exonuclease III and endonuclease IV: xth nfo
) are very sensitive to NO.
). These results suggest that, upon exposure of cells to NO.
, BER is required to process AP sites that are formed either by DNA glycosylases or by spontaneous base loss.
In addition to AP endonucleases, recombinational repair plays a pivotal role in preventing the genotoxic effects of NO.
). Homologous recombinational repair processes can be initiated by DNA double-strand breaks (DSBs) or single-stranded DNA regions (17
). In E. coli
, the RecBCD complex processes DSBs (1
), while the RecFOR proteins bind single-stranded DNA regions and facilitate resumption of replication (9
). To survive NO.
toxicity, E. coli
is dependent on RecBCD, but not on RecF, which suggests that the requirement for homologous recombination following NO.
exposure is not due to lesions that induce single-stranded regions or lesions that inhibit the replication fork but rather to the formation of DSBs (34
). As neither NO.
, nor peroxynitrite efficiently creates DSBs by direct reaction with DNA in vitro (4
), it is likely that NO.
induces other types of damage that are subsequently converted into DSBs by enzymatic processing and/or DNA replication.
DNA damage created by the exposure of cells to NO.
consists mostly of base damage (34
). Although some single-strand breaks appear immediately after exposure, the majority of single-strand breaks do not appear until hours after NO.
exposure (at least in Chinese hamster ovary cells) (37
). This delayed appearance suggests that the majority of the single-strand breaks are created enzymatically. We therefore hypothesized that NO.
-induced recombination may be stimulated by the action of DNA glycosylases. NO.
induces DNA base damage that is removed by DNA glycosylases, thereby protecting the cell against mutations. However, the processing of DNA base damage by DNA glycosylases leads to the creation of AP sites and single-strand breaks. In their turn, AP sites and single-strand breaks may be converted into DSBs, possibly during DNA replication. Such DSBs can induce genetic rearrangements and make the cell dependent on recombinational repair.
In this work, we show that disruption of the DNA glycosylase Ung or Fpg rescues AP endonuclease-deficient E. coli
toxicity. We infer that these two DNA glycosylases are active in the production of AP sites, which are toxic to an AP endonuclease-deficient cell. Furthermore, we show that upon exposure of E. coli
these DNA glycosylases lead to a dependence on RecBCD, indicating DSB repair. While overexpression of DNA glycosylases has been shown previously to sensitize recombinational repair-deficient E. coli
), here we show that cells with normal expression levels of DNA glycosylases are sensitive to NO.
-induced DNA DSBs. The results presented in this work shed light on the effects of DNA glycosylases on the maintenance of genomic stability. While DNA glycosylases may protect against NO.
-induced mutations, they simultaneously put cells at risk of genetic rearrangements.