DNA damage promotes cancer, aging, neurological disorders and heritable diseases. Exposure to DNA damage is unavoidable, as DNA damaging agents are ubiquitous both in our environment and within our cells. Despite the importance of DNA damage, technological obstacles limit routine measurements of DNA damage. We have recently collaborated with materials scientists and engineers to develop the "CometChip", a new technology for measuring DNA damage that has its basis in the well-accepted Comet assay. Here, we propose to leverage our technology toward translation by performing studies that are essential in order to enable the application of this technology to studies of DNA repair capacity in people. We have demonstrated that the CometChip is effective for measuring both DNA damage and DNA repair in human white blood cells in vitro and have developed image analysis software so that samples can be analyzed in an automated fashion, which greatly increases throughput and makes the assay more robust. We are excited about the potential utility of the CometChip for a broad range of applications. Of particular interest is the possibility of identifying people who are born with lower than average levels of DNA repair, since these people would want to take special precautions to prevent cancer and may need special consideration with regard to pharmaceuticals. In order to optimize our platform for identifying people with DNA repair deficits, we first need to work with cell lines that carry known DNA repair deficiencies. Ideally, we would like to compare cells that are the same genetically with the exception of suppressed expression of a single DNA repair gene. To accomplish this, we have knocked down expression of DNA repair genes to create isogenic cell lines harbouring specific deficiencies. Here we propose to complete creation of a set of 10 knock down cell lines in which two of the key genes for each of five major DNA repair pathways are knocked down. We propose to optimize conditions and parameters of the CometChip to reveal the presence of these defects. Importantly, the assay will be optimized for analysis of primary human cells compatible with population studies. Our hypothesis is that by varying the quality and quantity of the DNA damage, as well as the conditions of analysis, we will be able to identify deficiencies in at least four major DNA repair pathways in parallel and reveal variations in the balance among DNA repair pathways. Resulting data will shed new light on how our genes affect susceptibility to environmental exposures, and in turn, how exposures can affect the levels of DNA damage in people. We anticipate that the proposed CometChip platform will have broad applications in industry, academia, medicine and public health, thus enabling the application of tools and understanding from basic research to directly benefit the public.
We propose to develop a high throughput DNA damage analysis platform for studies of DNA damage in human cells. The new capabilities that we will develop will have many applications, including drug discovery and genotoxicity testing (important for the pharmaceutical industry), detection of DNA repair deficiencies among people and among cells (important for personalized medicine), and detection of increased levels of DNA damage among people exposed to environmental hazards (important for public health). Taken together, the proposed studies will greatly accelerate translation of a highly valuable technology.
|Fang, Qingming; Inanc, Burcu; Schamus, Sandy et al. (2014) HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase ?. Nat Commun 5:5513|
|Choi, Yong Jun; Li, Han; Son, Mi Young et al. (2014) Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair. PLoS One 9:e86358|
|Fouquerel, Elise; Goellner, Eva M; Yu, Zhongxun et al. (2014) ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion. Cell Rep 8:1819-31|
|Ge, Jing; Prasongtanakij, Somsak; Wood, David K et al. (2014) CometChip: a high-throughput 96-well platform for measuring DNA damage in microarrayed human cells. J Vis Exp :e50607|
|Lan, Li; Nakajima, Satoshi; Wei, Leizhen et al. (2014) Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin. Nucleic Acids Res 42:2330-45|
|Weingeist, David M; Ge, Jing; Wood, David K et al. (2013) Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors. Cell Cycle 12:907-15|
|Ge, Jing; Wood, David K; Weingeist, David M et al. (2013) Standard fluorescent imaging of live cells is highly genotoxic. Cytometry A 83:552-60|
|Mutamba, James T; Svilar, David; Prasongtanakij, Somsak et al. (2011) XRCC1 and base excision repair balance in response to nitric oxide. DNA Repair (Amst) 10:1282-93|