Real-time Base Excision Repair Assay to Measure the Activity of the 8-oxoguanine DNA Glycosylase 1 in Isolated Mitochondria of Human Skin Fibroblasts.

Abstract

7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common and mutagenic oxidative DNA damages induced by reactive oxygen species (ROS). Since ROS is mainly produced in the inner membranes of the mitochondria, these organelles and especially the mitochondrial DNA (mtDNA) contained therein are particularly affected by this damage. Insufficient elimination of 8-oxoG can lead to mutations and thus to severe mitochondrial dysfunctions. To eliminate 8-oxoG, the human body uses the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which is the main antagonist to oxidative damage to DNA. However, previous work suggests that the activity of the human OGG1 (hOGG1) decreases with age, leading to an age-related accumulation of 8-oxoG. A better understanding of the exact mechanisms of hOGG1 could lead to the discovery of new targets and thus be of great importance for the development of preventive therapies. Because of this, we developed a real-time base excision repair assay with a specially designed double-stranded reporter oligonucleotides to measure the activity of hOGG1 in lysates of isolated mitochondria. This system presented here differs from the classical assays, in which an endpoint determination is performed via a denaturing acrylamide gel, by the possibility to measure the hOGG1 activity in real-time. In addition, to determine the activity of each enzymatic step (N-glycosylase and AP-lyase activity) of this bifunctional enzyme, a melting curve analysis can also be performed. After isolation of mitochondria from human fibroblasts using various centrifugation steps, they are lysed and then incubated with specially designed reporter oligonucleotides. The subsequent measurement of hOGG1 activity is performed in a conventional real-time PCR system.