Environmental Factor, July 2008, National Institute of Environmental Health Sciences
Team Develops Novel Method to Monitor DNA Damage Recognition
By Senyene Hunter
In a study published in the June issue of Nanoletters, Principal Investigator Bennett Van Houten, Ph.D., and Postdoctoral Fellow Hong Wang, Ph.D., in the NIEHS Laboratory of Molecular Genetics DNA Repair and Mitochondrial Damage Group, along with their collaborators from the University of North Carolina, present novel methods that overcome obstacles encountered in single-molecule studies. The method involves the application of nanotechnology using quantum dots to characterize proteins.
Quantum dots are nanometer scaled fluorescent probes that have the advantage of being highly photostable, while displaying strong emission intensities. Using nanoparticles such as quantum dots to label and monitor the behavior of individual proteins is a relatively new development that could greatly advance biochemical research, particularly in the area of protein-DNA interactions. However, certain challenges have thus far restricted the use and effectiveness of these techniques. In the past, the use of quantum dot-conjugated proteins was limited by the inability to reliably determine the number of proteins attached to each quantum dot and the retained functionality of the conjugated proteins.
To overcome this problem, Wang and colleagues (https://www.ncbi.nlm.nih.gov/pubmed/18444686?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum) attached quantum dots to the protein UvrB using an antibody-sandwich method. UvrB is a bacterial DNA damage recognition protein involved in nucleotide excision repair. In this method, an epitope tag was attached to UvrB and antibodies were used as "adaptors" to connect the quantum dot to the tagged UvrB protein. Wang and colleagues then used atomic force microscopy (AFM) imaging to demonstrate the successful conjugation of a single quantum dot to one UvrB molecule, conclusively demonstrating a 1:1 stoichiometry. As Wang explained, AFM is a tool used for studying single molecule biomolecular interactions that "works in the same way as our fingers, which can touch and probe the environment. The 'finger' in AFM is a sharp tip which has a radius at nanometer scale."
The researchers also used AFM to identify interactions between quantum dot-conjugated UvrB (UvrB-QD) and its binding partner UvrA. These analyses indicated that UvrB-QD was still functional and that the presence of the quantum dot did not disrupt protein-protein interactions. The team was also able to use AFM imaging to visualize UvrB-QD conjugate loaded on to a DNA substrate containing damage by UvrA.
To verify results obtained via AFM imaging, Wang employed an agarose gel-based electrophoresis mobility shift assay (EMSA). EMSA is a commonly used and widely accepted method of evaluating the nucleic acid binding function of a protein. The investigators used EMSA analysis to demonstrate that "the UvrB-QD conjugate remains functional for DNA damage recognition and can be loaded specifically onto damaged DNA by UvrA."
Findings discussed in the paper have a wide range of applications. "These methods can be applied to any protein that has an epitope tag. In addition, these advancements will help further the use of quantum dot-labeled proteins in living cells to study intracellular processes," said Wang. Van Houten added, "Our long term goal is to use epi-fluorescence microscopy to follow these proteins in real time as they 'walk along' DNA in search of DNA damage. Ultimately we want to be able to visualize DNA repair, molecule by molecule, as they assemble on the site of damage and remove the DNA adduct."
Citation: Wang H, Tessmer I, Croteau DL, Erie DA, Van Houten B (https://www.ncbi.nlm.nih.gov/pubmed/18444686?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum) . 2008. Functional characterization and atomic force microscopy of a DNA repair protein conjugated to a quantum dot. Nano Lett 8(6):1631-1637.
(Senyene Hunter, Ph.D., is a postdoctoral fellow in the NIEHS DNA Repair and Mitochondrial Damage Group.)