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2002
LSU-HHMI Summer Undergraduate Research Program |
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 Melissa
Bueche (Huangen Ding, LSU Dept. of Biological Sciences)
Modification of DNA Repair Enzyme, Endonuclease III [4Fe-4S]
cluster, by Nitric Oxide and Peroxynitrite
Endonuclease III (Nth) is very important in repairing DNA oxidative
damage. It is highly conserved from bacteria to humans. In E.
coli, the cell cannot survive under oxidative stress without
endonuclease III. The goal of this study is to determine if
the [4Fe-4S] could be modified by reactive free radicals in
vitro. We treated purified endonuclease III with nitric oxide
(NO), and peroxynitrite. These two free radicals are produced
physiologically by activated macrophages at high concentrations.
Two different indicators were used to detect iron release from
the [4Fe-4S] cluster of endonuclease III. They are 2’,
2’ Dipyridyl (used to detect ferrous iron) and, Deferoxamine
(used to detect ferric iron). The results showed that significant
amounts of iron were released from purified endonuclease III
[4Fe-4S] cluster in the ferrous form when the protein was treated
with nitric oxide and peroxynitrite. Peroxynitrite seemed to
be more effective than nitric oxide in the amount of iron that
was released. The release of iron form the iron sulfur cluster
by nitric oxide and peroxynitrite was largely prevented by glutathione,
a tripeptide that contains a thiol group. It is likely that
glutathione competes with the free radicals, and as a result
less iron was released when endonuclease III was treated with
both free radicals. We also found that hydrogen peroxide released
small amounts of iron in the ferric form, but it is much less
effective in the release of iron form the endonuclease III [4Fe-4S]
cluster.
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