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Related Experiment Videos

Superoxide accelerates DNA damage by elevating free-iron levels

K Keyer1, J A Imlay

  • 1Department of Microbiology, University of Illinois, Urbana 61801, USA.

Proceedings of the National Academy of Sciences of the United States of America
|November 26, 1996
PubMed
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Superoxide causes DNA damage by releasing iron from iron-sulfur clusters, not storage proteins. This free iron then catalyzes harmful reactions, leading to mutagenesis and cell damage.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Superoxide is known to cause DNA damage, primarily through hydroxyl radical formation.
  • Previous hypotheses suggested superoxide-mediated DNA damage occurred via direct electron transfer to iron on DNA.
  • Recent studies have challenged this, proposing indirect mechanisms involving iron release.

Purpose of the Study:

  • To investigate the mechanism by which superoxide leads to oxidative DNA damage.
  • To determine the source of free iron that catalyzes DNA damage in the presence of superoxide.
  • To elucidate the role of iron storage proteins and iron-sulfur clusters in this process.

Main Methods:

  • Whole-cell electron paramagnetic resonance (EPR) spectroscopy to measure intracellular free iron levels.

Related Experiment Videos

  • Analysis of superoxide-stressed Escherichia coli, including wild-type and mutant strains lacking iron storage proteins.
  • Overexpression of an enzyme containing labile [4Fe-4S] clusters.
  • Assessment of spontaneous mutagenesis and DNA damage induced by hydrogen peroxide (H2O2).

    • Main Results:

    • Superoxide stress significantly increased intracellular levels of loose iron compared to unstressed cells.
    • Iron storage proteins were not the primary source of free iron; mutants lacking these proteins still showed increased iron.
    • Overproduction of an enzyme with labile [4Fe-4S] clusters markedly elevated free iron in cells.
    • Increased free iron correlated with higher rates of spontaneous mutagenesis and DNA damage from H2O2.
    • Growth defects and DNA damage induced by superoxide were linked to the damage of specific iron-sulfur clusters.

    Conclusions:

    • Superoxide promotes oxidative DNA damage by leaching iron from cellular iron-sulfur clusters, not primarily from storage proteins.
    • The released iron catalyzes the formation of DNA-damaging oxidants.
    • Damage to iron-sulfur clusters is a critical event underlying superoxide-induced cellular dysfunction and DNA damage.