Abstract
DNA base damage is generated continuously by various factors including oxidization, alkylation, and deamination. Reactive oxygen species (ROS) are generated as by-products of oxygen respiration in mitochondria, and also generated after exposure to radiation or ultraviolet. ROS induced oxidative DNA base damage leads to inhibition of replication, transcription, and mutation. Finally, those mutations induce cancer, aging, and neurological disorders. These damages are repaired primarily by base excision repair (BER) pathway. Endonuclease VIII-like 1 (NEIL1) possesses intrinsic AP lyase activity and cleave the DNA strand at the both sides of the apurinic (AP) site leaving a single nucleotide gap. Mouse NEIL1 has been reported to have two variants in mouse breast tumor or, aorta and vein but their physiological roles are unknown. We postulated the former as variant1, the latter as variant2. Previously, we have confirmed both variants' presence in normal tissue by RT-PCR. In this report, we initially extracted total RNA from each tissue of 8-week old male mouse followed by reverse transcription. Each primer sets recognizing NEIL1, variant1 and variant2, variant2 are designed for real time PCR. Beta-actin is used for internal standard of amount of mRNA. Because N-terminal tag fused NEIL1 had reported to lose its activity, we have examined the activity of C-terminal tag fused protein. Tag-fused variants had no activity. In this report, we attempted to assemble tag-free protein expression system to confirm the activity of purified protein toward damaged oligonucleotide.
