Background

The human body represents a permanent case of damage. Each of the approximately 10¹³ body cells suffers tens of thousands to about 100,000 damages to its DNA per day (Janion, 2001; Jackson and Bartek, 2009). 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common oxidative DNA damages induced by reactive oxygen species (ROS). By oxidizing the base guanine to 7,8-dihydro-8-oxoguanine, 8-oxoG, in contrast to guanine, is now able to hybridize not only with cytosine but also with adenine. This can lead to a complete base exchange after the second replication and thus trigger serious mutations. The replacement of a guanine/cytosine base pair by thymine/adenine is one of the most frequent mutations in human cancer. This so-called transversion also occurs frequently in the tumor suppressor gene p53 (Hirano, 2008). For this reason, and based on the fact that between 1,000 and 2,000 8-oxoG lesions per day occur in a healthy cell, while in tumor cells the number of 8-oxoG lesions increases up to 1,000,000, 8-oxoG is considered to be one of the major causes of cancer (Ravanat et al., 2000; Nakabeppu, 2014). Furthermore, 8-oxoG is associated with aging processes and degenerative diseases (such as Alzheimer's disease and Parkinson's disease) (Sampath et al., 2012; Nakabeppu, 2014) and thus shows great pathophysiological relevance.

To counteract these processes, the human body uses the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which is the main antagonist to oxidative damage to DNA. Before replication, it removes 8-oxoG by means of base excision repair (BER), replaces it with a guanine base and thus prevents the development of mutations (Radicella et al., 1997; Hamann et al., 2009). However, previous studies indicate that the activities of glycosylases and thus also the activity of the human OGG1 (hOGG1) decrease with increasing age (Loft and Poulsen, 1996; Gorbunova et al., 2007). This has the effect on an organism that sooner or later, as a result of the reduced hOGG1 activity and the resulting decrease in the ability to perform BER processes, an age-related accumulation of 8-oxoG occurs. 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.

For the measurement of the hOGG1 activity we developed an assay that exploits the principle of enzymatic removal of 8-oxoG from DNA as well as the cut into its sugar-phosphate backbone by hOGG1 as a part of the BER. The principle of the enzymatic removal of 8-oxoG from DNA as well as the incision in its sugar-phosphate backbone by hOGG1 during a BER is used. Lysates from isolated mitochondria are incubated with specially designed reporter oligonucleotides for this assay. Each of the two double-stranded constructs consists of 33 nucleotides and identical sequence. In addition, both carry a 6-carboxyfluorescein (6-FAM) molecule at each end of the reporter strand and a black hole quencher (BHQ) at each end of the complementary strand (Figure 1). Thus, in each double-stranded construct, the 6-FAM molecule (fluorescent dye) is opposite of BHQ which suppresses the fluorescence of the dye. The DNA glycosylase present in the lysates from the mitochondria recognizes the damage to the reporter oligonucleotide (CN-8-oxoG) and first cuts out the oxidized base, followed by a cut in the phosphate backbone. The incised DNA strand then dissociates from the counter strand at the incubation temperature of 37 °C and a fluorescence signal can be measured. The strength of the signal correlates with the repair activity or AP lyase activity of the hOGG1 enzyme. The use of an undamaged reporter oligonucleotide (CN-CTRL) serves as a control to exclude unspecific digestion of the oligonucleotides (Figure 2A). To determine the resulting oligonucleotide fragments, a melting curve analysis is performed at a temperature of 95 °C to 20 °C after completion of the real-time activity measurement. For this purpose, the first derivation of the trend of the fluorescence signal as a function of temperature is recorded (-(d/dT)). The resulting minima represent the melting temperatures of the different fragments (Figure 2B).