DNA Mediated Charge Transport in p53

CHM 4390-U02
Rohit Vinod
April 22, 2015
INTRODUCTION
Human transcription factor p53 is considered to be an important and useful protein that plays a key role in cancer and tumor suppression. Many of the pathways in which p53 is utilized usually are apoptosis, cell deterioration also known as senescence, cell cycle arrest or DNA repair (Riley et. al, 2008). P53 also plays a role in glucose metabolism as a regulator of glycolytic and oxidative phosphorylation through positive and negative regulation of certain genes and the proteins that they encode for (Madan et. al, 2011). Usually if there are mutations in the genes that eventually translate into p53 then there are significant chance that tumors start to develop as seen in more than half of human cancers. In addition to such necessary roles, p53 also can sense oxidative stress and binds to redox-active DNA which often receives oxidative damage due to election migration through the DNA base stack. Many other sources of DNA oxidation can arise as a result of ionizing radiation, exogenous chemicals and even metabolic side products (Generaux et. al, 2010). DNA can be utilized in charge transport over distances of 100 base pairs or 34 nm in length (Augustyn et. al, 2013). The significance of the DNA oxidation and p53’s ability to sense oxidative stress is essential in understanding how DNA can be utilized in charge transport. In particular, redox activation in p53 occurs using DNA CT and the thiol switches used (Sontz, 2007). P53 was examined due to the protein having 10 cysteine residues where cysteine is often used in vivo by DNA-bound proteins for its redox capabilities. The experiment conducted by Schaefer and Barton (2014) examines how DNA sequence plays in greater detail in terms of selectivity and sequence context. The experimenters hypothesized that guanine doublets and triplets would affect the ability of DNA CT. The prediction for this experiment was to see if more guanine doublets or triplets could result in higher oxidative damage in p53 protein.

METHODS There are several steps at which the researchers used to help collect the data they were seeking. The oligonucleotides used in the experiment were synthesized on an ABI 3400 DNA synthesizer. There were two different groups of sequences that were synthesized, ones containing anthraquinone (AQ) and that have the dimethoxytrityl (DMT) group. The protein that was examined had three stabilizing mutations: M133L, V203A, and N268D with the gene for p53’ coming from a quadruple-mutated plasmid with N239Y/M133L/V203A/N268D to ensure that under high conditions of radiation and temperature the protein does not denature. The oligonucleotides were radiolabeled in 5’ end for single stranded and the 3’ end for DNA strands conjugated with AQ. The oligonucleotides were then allowed to bind to the p53’ in a 1:1 ratio with the concentration of p53’ per sequence being determined using KD of the protein with natural consensus sequences to ensure 80% of DNA is bound to the protein. After the DNA binds with the protein, radioactivity of each sample was used by scintillation counting by a Beckman LS 5000TD and normalized and then the DNA was loaded into polyacrylamide gel and buffer with the blots being recorded and then exposed to a phosphorimaging screen. The oxidative DNA damage was assayed by passing samples through radiation fir 1 hour to ensure that oxidative damage can be properly examined. Afterward there was a gel shift assay for four synthetic consensus assay to see how dissociation occurs based on upon DNA sequence over time of exposure to radiation. The sequences are labeled as AAA, AGG, GGG, and GGG/GGG. Also dissociation was examined in natural types of p53 response elements such as caspase and s100A following exposure to radiation and compare them to the synthetic sequences. Finally, they examined oxidative damage with and without the protein using denaturing polyacrylamide gel.
DISCUSSION
The results from the procedures that took place all correspond to a full length p53 protein with three thermodynamically stable mutations which are M133L, V203A and N268D. The key thing to note in relation to the results is that the mutations do not provide major differences in DNA binding ability or in oxidative association when compared to the wild-type protein but at the same time allows for a strong and stable protein that is necessary for experimentation (Schaefer and Barton, 2014). The independent variable was the four different sequences of AAA, AGG, GGG, and GGG/GGG with guanine being directly examined because of the electron and electron hole migration through Π-bond stacking. This is because electron holes in DNA localize to low redox potential which was observed mostly in guanine doublets and triplets. One observation that can be made is that oxidative damage is apparent at the 5’ G of guanine doublets and triplets as well as tethered oxidants of an AQ-AAA sequence, although minimally, which is not contained in the consensus sequence of the p53 gene. Other information that can be drawn from these experiments is that locations of low redox potential should align with the p53-DNA groove interface to allow effective DNA-protein electron transfer. The importance of these findings allows for effective DNA CT because it can create better understanding as how DNA charge transport can be used for DNA repair and signal transfer. P53’ dissociation was highest at AQ-GGG which was at 22.3% and the AQ-GGG/GGG had around 13.0% with the AQ-AGG being in the middle (Schaefer and Barton, 2014). AQ-GGG has two locations in which holes can reside while AQ-GGG/GGG has four with the density is lower in the sequence with the two sets of guanine triplets. Moreover, p53 which is supposed to inhibit oxidative damage is only able to if the particular part of the sequence is directly aligned to the protein. When establishing similarities between the natural binding sites versus synthetic sites it was shown that S100A produces a similar response compared to the sequence containing two guanine triplets which would result in diminished tumor suppressor activity while Caspase operates similar to the synthetic adenine triplet examined meaning there was minimal dissociation There may be a concern that would need to be addressed if there were no similarities being made between the natural and synthetic binding sites to establish how in vivo processes differ from ones observed in vitro. The significance of this experiment was to examine how DNA Charge Transport can affect the viability of a cell and the pathways that can be affected as a result of DNA CT occurring. Also, the research discussed in the article also served to examine a possible pathway in which human tumors can be produced through DNA CT in one tumor suppressor protein.

REFERENCES
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