Lee, Paul Eulehwann (2010) Activation of transcription from a distance: investigations on the oxidation of SoxR by DNA-Mediated charge transport. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:03192010-175848794
In enteric bacteria, the cellular response to oxidative stress caused by superoxide is activated by soxR, which encodes a redox-active transcription factor that contains a [2Fe2S] cluster and binds DNA with high affinity. Here we describe how SoxR may detect global changes in oxidative stress while bound to DNA at a single location through DNA-mediated charge transport. A unique property of DNA is its ability to delocalize charge along its base stack, allowing oxidative damage to be funneled to specific sites of low oxidation potential. Charge transport also has the potential to access proteins with redox-active moieties. Electrochemical studies presented here demonstrate that the redox couple of the [2Fe2S] clusters of SoxR can be accessed through the DNA, and that when the protein is bound to DNA, is shifted almost 0.5 V positive to its potential measured in solution in the absence of DNA. SoxR in its reduced form is found to inhibit guanine damage by repairing guanine radicals formed in DNA by the use of various photoactive metallointercalators, by donating an electron from one of its [2Fe2S]+ clusters and filling the guanine radical hole. RT-PCR is used to monitor the amount of soxS mRNA produced in cells that have taken up the DNA binding photooxidant [Rh(phi)2bpy]3+ and are treated with light. Cells thus treated to generate guanine radicals express soxS, evidence that SoxR is being oxidized. An in vitro assay is furthermore used to examine directly the DNA-mediated oxidation of SoxR by measuring its transcriptional activity. [Rh(phi)2bpy’]3+, tethered to DNA 80 bp from the soxS promoter, induces transcription by activating SoxR upon irradiation. These results demonstrate not only that guanine radicals can act to oxidize SoxR, but that the resulting oxidized, DNA-bound protein is biologically active. Thus, transcription can be activated from a distance through DNA-mediated charge transport. The ability of DNA to conduct charge along its base stack allows offers a general strategy for DNA-mediated signaling of oxidative stress, as it allows information about oxidative events to be transmitted quickly and directly to the proteins responsible for turning on the genes necessary for cell survival.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||DNA; SoxR; DNA-mediated; transcription; oxidative stress|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||21 December 2009|
|Non-Caltech Author Email:||pelee1 (AT) gmail.com|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Paul Lee|
|Deposited On:||05 Apr 2010 18:38|
|Last Modified:||22 Aug 2016 21:18|
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