Currently, there have been at least 15 DNA polymerases reported in eucaryotic cells, the roles of which have not all been determined. Multiple DNA polymerases within cells allow the evolution of different polymerases for special or unique functions. Although some cross over between polymerases, they can be classified into 3 main groups, Replicative, Repair, and Specialized. The main role of replicative polymerases is to duplicate the organism’s genome while another set of polymerases are mainly involved in DNA repair. The final set is hypothesized to have specialized functions such as the bypass of DNA damage or translesion synthesis (TLS). Current models of TLS during cellular DNA replication hypothesize that DNA polymerase switching occurs in order to replace a replicative polymerase that is stalled or inhibited by a DNA lesion with a specialized DNA polymerase or set of polymerases that are more facile at performing TLS. Once the lesion is bypassed, the specialized polymerase is then replaced by the replicative polymerase and normal replication is resumed. This process of switching may result in mutations being incorporated and could be important in the choice of chemotherapy that is given to cancer patients, since may drug therapies cause DNA damage.
I am interested in approaching this area of research by several different angles. The first set of experiments would include in vitro (in the test tube) studies to examine the interactions between polymerases and the DNA template which can be examined through the addition of a fluorophore to the polymerase and a dark quencher placed on the 3’ end of the DNA template. The combined use of fluorescent markers and quenchers will allow detection of a bound polymerase to the template DNA as a loss of fluorescence due to fluorescence resonance energy transfer (FRET).
The second line of research I would like to continue my current research on regulation of polymerase levels and their affect on DNA replication and stability. This line of research would be include ex vivo experiments in human cell lines and would include both normal and tumorigenic cells lines. Our initial results indicate that polymerase levels may be altered following treatment of cell lines with chemotherapeutics. DNA array technology, along with Northern and Western blot analysis would play a role to determine the pathways involved in polymerase regulation.
I would also like to extend my research into a vertebrate model, zebrafish. The zebrafish (Danio rerio) animal model was chosen for this research for several reasons. First, the zebrafish is a 3 - 4 cm, hardy, freshwater vertebrate animal and therefore the results obtained from this project are more easily extrapolated to people. A single pair of zebrafish can produce hundreds of eggs biweekly with a generation time of 3 months. The embryos, about 1 mm in diameter, develop ex utero (outside the body) and are completely transparent allowing visualization of the animal’s internal structures during development. The final line of proposed research would include tissue-specific and temporal expression of DNA polymerases in developing zebrafish may help elucidate their function and may easily be completed through RNA and protein analysis. The functions of DNA polymerases can be studied in vivo (in the animal) using current techniques such as morpholino technology and microinjection into fertilized eggs.