| Oxidative DNA damage due to reactive oxygen species (ROS) can cause single-base alterations such as 8-oxo-7,8-dihydroguanine (8oxodG) lesions, which are repaired by the base excision DNA repair (BER) pathway. Assays developed to date, to measure BER, are limited by in vitro analysis and use of radiolabeled reagents. This thesis outlines the design of a novel assay to measure the kinetics of BER in vivo, using a biotin-tagged oligonucleotide DNA substrate that contains an 8oxodG lesion. The substrate DNA is transfected into target cells and incubated to allow repair of the lesions. The DNA is then captured following cell lysis and the disappearance of 8oxodG lesions is monitored using competitive 8oxodG ELISA. This assay was developed to corroborate the results obtained from our investigations on the role of the breast cancer susceptibility protein-1 (BRCA1) in BER, using another novel in vivo assay called the host-cell reactivation (HCR) assay. Preliminary results from this study showed that BRCA1 is involved in BER. Together, these in vivo assays may be used to identify potential cancer genes that regulate the BER pathway. |
