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. 1996 Nov 26;93(24):13635-40.
doi: 10.1073/pnas.93.24.13635.

"V体育安卓版" Superoxide accelerates DNA damage by elevating free-iron levels

K Keyer  1 , J A Imlay
Affiliations

VSports app下载 - Superoxide accelerates DNA damage by elevating free-iron levels

K Keyer et al. Proc Natl Acad Sci U S A. .

Abstract

Superoxide promotes hydroxyl-radical formation and consequent DNA damage in cells of all types. The long-standing hypothesis that it primarily does so by delivering electrons to adventitious iron on DNA was refuted by recent studies in Escherichia coli. Alternative proposals have suggested that superoxide may accelerate oxidative DNA damage by leaching iron from storage proteins or enzymic [4Fe-4S] clusters. The released iron might then deposit on the surface of the DNA, where it could catalyze the formation of DNA oxidants using other electron donors. The latter model is affirmed by the experiments described here. Whole-cell electron paramagnetic resonance demonstrated that the level of loose iron in superoxide-stressed cells greatly exceeds that of unstressed cells. Bacterial iron storage proteins were not the major source for free iron, since superoxide also increased iron levels in mutants lacking these iron storage proteins. However, overproduction of an enzyme containing a labile [4Fe-4S] cluster dramatically increased the free iron content of cells when they were growing in air. The rates of spontaneous mutagenesis and DNA damage from exogenous H2O2 increased commensurately. It is striking that both growth defects and DNA damage caused by superoxide ensue from its ability to damage a subset of iron-sulfur clusters. VSports手机版.

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Figures

Figure 1
Figure 1
Iron EPR signals from whole-cell preparations. EPR parameters were: T = −125°C, field center = 1520 G, field sweep = 500 G, modulation amplitude = 25 G, receiver gain = 32,000, frequency = 9.006 GHz, and power = 30 mW. One thousand data points were collected per scan, and four scans were averaged per sample. Scan profiles for samples B–F have been normalized to cell concentration. Scans: A, iron standard; B, AB1157 (wild type), 10 μM intracellular chelatable iron; C, KK204 (Fur−), 70 μM iron; D, JI132 (SOD−), 80 μM iron; E, KK216 (SOD− Fur−), 160 μM iron; F, same AB1157 scan as B with 13-fold amplification of signal.
Figure 2
Figure 2
Effect of 6-phosphogluconate dehydratase overproduction or exposure to paraquat upon the EPR signals of SOD-proficient cells. Assay conditions were similar to those described in Fig. 1, except: power = 50 mW, modulation amplitude = 16 G, and receiver gain = 20,000. Scans: A, wild-type cells (AB1157), 10 μM intracellular free iron; B, 6-phosphogluconate dehydratase overproducer (KK230), 35 μM iron; C, AB1157 during exposure to 200 μM paraquat, 40 μM iron. This concentration of paraquat was just sufficient to reduce the rate of growth by about 50%.
Figure 3
Figure 3
Relationship between internal iron concentration and sensitivity to killing by H2O2. Cell concentrates of the congenic strains AB1157 (wild type), JI132 (SOD−), KK204 (Fur−), KK206 (bfr ftn), KK209 (SOD- bfr ftn), KK216 (SOD− Fur−), KK230 (SOD+ Edd overproducer), and KK231 (SOD− Edd overproducer) were grown aerobically and assayed for internal iron by EPR. The sensitivity of the exponentially growing cultures to killing by exogenous H2O2 was determined after 8 min of exposure to 2.5 mM H2O2 in the same medium (8); other work has shown that the consequent cell death is due to iron-catalyzed DNA damage (52). Killing rates [-Ln(surviving fraction)/8] have been normalized to that of JI132 (8% survival). Error bars signify standard deviations of at least four independent experiments.
Figure 4
Figure 4
Aerobic and anaerobic mutagenesis using trimethoprim. Error bars represent standard deviations from at least three experiments.
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