This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. DNA encodes the essential information required for cellular activities, and damage to this genetic material is the driving force in cancer and aging in humans. DNA damage is caused spontaneously by byproducts of cellular metabolism, as well as by exogenous agents such as ionizing radiation and environmental chemicals. Estimates of the amount of oxidative DNA damage each cell in the body experiences are on the order of ~10^5 events per day. Humans possess multiple systems of proteins that remove damaged nucleotides and replace them in a process called excision repair which includes incorporation of new nucleotides into the DNA. Current techniques are insufficiently sensitive to measure this repair synthesis that occurs as part of normal cellular metabolism. In this proposal, we will employ the very sensitive measurement capabilities of accelerator mass spectrometry (AMS) to quantify spontaneous DNA repair by measuring the amount of incorporated 14C-labeled thymidine (one of the four DNA bases). We will also measure repair synthesis in cells after exposures to very low doses of DNA-damaging agents that are directly relevant to human health. Since the initiation of this study, we have successfully determined the experimental parameters necessary for control of cell growth and 14C thymidine labeling. Cells progressing through S-phase are readily detectable and thymidine quantification is consistent with expected levels of incorporation during synthesis. Preliminary results in small pools of cells without contaminating S-phase cells suggest the possibility of measuring spontaneous thymidine incorporation in single cells. This level of sensitivity may allow quantification of repair, induced by very low levels of DNA damaging agents, at single cell resolution, enabling us to determine cell-to-cell variability. Results from these proof-of-principle studies are expected to provide a foundation for future work in which cellular parameters that govern rates of oxidative damage and repair are investigated in much greater detail.
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