期刊
BIOCHEMISTRY
卷 51, 期 33, 页码 6679-6689出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi300703x
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资金
- National Science Foundation [MCB-0744240, 1051610]
- Div Of Biological Infrastructure
- Direct For Biological Sciences [1051610] Funding Source: National Science Foundation
The prokaryotic DNA protection during starvation (Dps) proteins typically protect: macromolecules against damaging agents via physical association with DNA and by oxidizing and sequestering iron. However, Deinococcus radiodurans Dps-1, which binds DNA with high affinity, fails to protect DNA against hydroxyl radicals due to iron leakage from the core, raising the question of how (OH)-O-center dot-mediated damage to Dps-1-bound DNA is avoided. As shown here, Mn(II) inhibits ferroxidase activity, suggesting that ferroxidation may be prevented in vivo as D. radiodurans accumulates a high ratio of Mn:Fe. Dps-1 has an N-terminal extension with a unique metal-binding site, an extension that has been proposed to be important for DNA binding and dodecameric assembly. Electrophoretic mobility shift assays show that Mn(II) restores DNA binding to bipyridyl-treated Dps-1, whereas Fe(II) fails to do so in the presence of H2O2, thus preventing DNA binding under conditions of ongoing ferroxidase activity. We also show that disruption of the N-terminal metal site leads to a significant reduction in DNA binding and to compromised oligomeric assembly, with the mutant protein assembling into a hexamer in the presence of divalent metal. We propose that securing the N-terminal loop by metal binding is required to initiate dodecameric assembly by contacting the neighboring dimer and that the absence of such optimal contacts results in formation of a hexameric assembly intermediate in which three dimers associate about one of the 3-fold axes. Once dodecameric Dps-1 is assembled, metal binding no longer affects oligomeric state; instead, differential metal binding controls DNA interaction under conditions of oxidative stress.
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