Mitochondrial base excision repair assays

Abstract

Mitochondrial DNA (mtDNA) is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to be particularly deleterious in post-mitotic cells, including neurons, and to play a critical role in the aging process and in a variety of diseases. Thus, efficient mtDNA repair is important for the maintenance of genomic integrity and a healthy life. The base excision repair (BER) mechanism was the first to be described in mitochondria, and consequently it is the best known. This chapter outlines protocols for isolating mitochondria from mammalian cells in culture and from rodent tissues including liver and brain. It also covers the isolation of synaptic mitochondria. BER takes place in four distinct steps, and protocols describing in vitro assays for measuring these enzymatic steps in lysates of isolated mitochondria are included.