DNA polymerase 4 (Pol 4) is an essential enzyme which plays critical roles in both DNA replication and DNA repair. The enzyme consists of four subunits: p125 and p50 which make up the catalytic core, and p68 and p12, which provide the enzyme increased stability, enhanced activity, and additional sites for binding of other protein partners. Previous experiments discovered a novel DNA damage response in which p12, the smallest subunit of Pol 4, was rapidly degraded in response to DNA damage, thereby generating a three-subunit DNA polymerase 4 enzyme (Pol 43). Kinetic studies were performed in order to compare the enzymatic properties of Pol 4 and Pol 43. Interestingly, it was determined Pol 43 demonstrated enhanced fidelity and proofreading abilities, and increased stalling at DNA lesions. Thus, the degradation of p12 in response to DNA damage converts the Pol 4 enzyme into one which can better repair DNA lesions, indicating a potential functional reason for p12 degradation. The long term goals and specific aims of this project focus on obtaining more comprehensive understanding of p12 degradation. The degradation of p12 will be characterized in terms of time- and dose-response, and the p12 half-life for each specific agent in a given cell line will be determined. This will be done through Western blotting and densitometry. In addition, cell lines which are deficient in specific DNA damage response proteins (such as p53 or ATM) will be utilized, such that the signaling processes which lead to p12 degradation can be more comprehensively understood. Next, cells will be synchronized by serum starvation or counterflow centrifugal elutriation such that the levels of p12 can be assessed at various stages in the cell cycle. From there, the effects of siRNA depletion of p12 on DNA repair will be assessed by measuring the rate of DNA repair, through clearance of cyclobutane pyrimidine dimers and 3H2AX foci, measured by flow cytometry and immunofluorescence microscopy methods. It would be expected that when p12 is depleted, repair would occur more quickly. Complementary experiments in which p12 degradation is blocked with proteasomal inhibitors will also be utilized, and should be expected to generate the opposite result from p12 depletion experiments. Finally, a number of potential E3 ligase enzymes which may ubiquitinate p12 to signal its degradation will be analyzed by siRNA in order to determine the involvement of these enzyme in p12 degradation. The ubiquitination of p12 is still not well understood, and thus the identification of the E3 ligase involved in p12 degradation is incredibly important. This project maintains a strong health focus in that the functioning of the DNA damage response is vital to the maintenance of genomic integrity, and thus the prevention of many cancers and other diseases.
In order to prevent cancer and limit disease, human cells are continuously fighting to repair DNA damage by activation of the DNA damage response and through intricate DNA repair mechanisms. We plan to perform in- depth characterization of a newly discovered aspect of the DNA damage response, namely, the damage- induced degradation of a protein subunit, which thereby allows the parent enzyme to more easily identify and correct damaged DNA, thus likely providing an adaptive and functional reason for the observed subunit degradation. Through our complete characterization of the response, we will obtain further knowledge and understanding about this DNA damage response, which may provide important clues to DNA repair in many cancers and other diseases brought about by genetic instability. )