Predicting the Effects of Intracellular Protein Variation on Base Excision Repair Capacity in Human Cells

Base excision repair (BER) is one of the primary means by which cells cope with genotoxic stress and DNA damage. BER is carried out by a series of enzymes which excise a damaged base from a DNA strand and replace it with an undamaged base. The importance of BER to the maintenance of genomic integrity, cellular health, and the health of an individual cannot be overstated. Numerous diseases, particularly cancer, have been correlated to BER deficiencies in several ways. 1) Increased disease risk has been correlated to elevated levels of highly mutagenic lesions repaired exclusively by the BER pathway. Elevated tissue levels of reactive oxygen species (ROS) which generate such lesions has likewise been correlated to increased disease risk. 2) Some functional variants of BER enzymes have been shown to be correlated with elevated disease risk. 3) Tumor cells have been shown to express BER enzymes at altered levels compared to surrounding healthy tissue.;Although the correlation between BER deficiency and disease risk has been thoroughly demonstrated it has not been well characterized. An unrepaired DNA lesion can lead to mutagenesis. Yet all cells cope with thousands of potentially mutagenic lesions each day, the majority of which are repaired without incident. Simply stated, mutagenesis can occur when a cell faces a damage load which exceeds its repair capacity. Therefore the need to characterize the quantitative relationships that govern repair capacity is central to understanding the development of diseases triggered by mutagenesis.;To this end I have created a formal model of the BER pathway. To test the model, I established protocols for DNA damage measurement in cultured human cells using single cell gel electrophoresis (SCGE). I developed novel software for the quantitation of SCGE data. In order to measure the system response following a perturbation, I created three cell lines deficient in the critical BER enzyme polymerase beta (polbeta) using RNA interference (RNAi) on FIEK 293t cells. Po113 knockdown was confirmed and quantified with Western blotting. Repair curves were generated for wild type and knockdown cell lines using SCGE. Model validity was tested by comparing model predictions and experimental results.