Modelling the impact of cellular variation on flux through base excision DNA repair.

Base excision repair (BER) is a pathway that repairs DNA damage inflicted by oxidative stress, certain alkylating agents, and spontaneous hydrolysis. Assays to measure the BER pathway are usually limited by being low-throughput, time consuming, and unable to measure each step of the pathway in addition to complete repair. The aim of the present work was to develop an assay format to overcome these limitations, and use this to develop a mathematical model to examine differences between cell types on flux through the pathway. A novel assay format was developed and optimised to measure uracil DNA glycosylase, AP endonuclease, DNA polymerase β, DNA ligase, AP site repair and complete base repair. Data was generated for each enzyme activity in HepG2, Caco-2, and peripheral blood mononuclear cells (PBMCs), and the effects on the pathway were predicted using a mathematical model. In addition, the assay was used to examine variability in enzyme activity between the two cell lines, correlations between enzyme activities in primary human cells, and the effects of caloric restriction on BER in a human weight loss intervention trial. The model revealed marked differences in the response to aberrant uracil between the two immortalised cell lines and the PBMCs, with PBMC nuclear extract in general excising the base more slowly and causing a smaller and slower accumulation of harmful intermediates, such as abasic sites and single strand breaks, compared to the immortalised cell lines. The model underestimated complete repair when compared to the biological data for all cell types, potentially due to cooperativity between BER enzymes not captured when repair steps were measured individually under experimental conditions. There were also significant correlations found between enzyme activities in PBMC extracts from different individuals, and a significant predictive effect of weight loss method on polymerase β activity in the caloric restriction trial. In conclusion, the research described here uncovered novel information regarding the effects of weight loss on DNA repair, and correlations between BER enzyme activities in healthy volunteers. This is also the first work to compare BER profiles of different cell types using biological data and mathematical modelling