Optimizing an in vitro assay for double strand break repair activities in mitochondrial and nuclear extracts

Abstract

DNA is constantly exposed to damaging agents from both endogenous and exogenous sources. In the case of mitochondrial DNA (mtDNA), damage is thought to be more pronounced due to its physical proximity to the respiratory chain, where large amounts of reactive oxygen species (ROS) are generated. These species can react readily with both the bases as well as with the deoxyribose, resulting in oxidized bases and DNA single or double strand breaks. In the nucleus, to preserve genome integrity, DNA double strand breaks (DSBs) can be repaired by one of two distinct pathways: non homologous end joining (NHEJ) and homologous recombination (HR). While the NHEJ and HR pathways have been thoroughly characterized in the nucleus, our knowledge about the role of these mechanisms in mtDNA repair is still very limited. We have set out to characterize the proteins involved in DSB repair in human mitochondria. Our results so far have shown that canonical NHEJ (Ku, DNA-PKCs) and HR (ATM, Rad51, Rad52) proteins are found in human mitochondrial extracts, suggesting that these proteins could be responsible for the double strand break repair activities observed. To test this hypothesis, we have developed knockdown cell lines for each target gene (Rad51, Rad52, Ku, DNA-PKCs, ATM), which have been already validated. The next step is to use mitochondrial extracts from these cell lines in in vitro double strand break repair assay to check for lower activity when compared to extracts from wild type cells. To do that, we have tried to reproduce the in vitro assays published in the literature with mitochondrial extracts, however with very little success. In attempts to get the in vitro assay working, several modifications of the protocol were tried, however no ligation activity has been detected yet. Therefore, and given the importance of this functional assay for characterizing the role of each protein in mitochondrial double strand break repair, we propose this collaboration whitch has a main aim develop an in vitro assay to measure double strand break repair activities in mitochondrial and nuclear extracts from human cells with depleted expression of selected proteins involved in DSB repair. (AU)