人類鹼基切除修復因子 nth endonuclease III-like 1 之功能性探討

Abstract

Oxidative stress from environmental agents and endogenous metabolic processes is a common source to cause DNA modifications, which result in mutations and genomic instability. In mammalian cells, two major DNA glycosylases nth endonuclease III-like 1 (NTHL1) and 8-oxoguanine DNA glycosylase 1 (OGG1) remove oxidative DNA damage through the base excision repair (BER) pathway. Previous studies have found that OGG1 plays an important role in DNA repair and maintaining cell viability. But the role of NTHL1 in oxidative stress-induced DNA damage and repair was not extensively examined. To elucidate the role of NTHL1 in oxidative stress-induced DNA repair and to know whether NTHL1 and OGG1 have synergistic effects with each other in DNA repair, we established stable nthl1, ogg1, and nthl1/ogg1 double knock-down (KD) cells by using lentivirus-based shRNA system. Using MTT assays, we found that all the KD cells examined were sensitive to oxidative stress, suggesting that NTHL1 is required for repairing oxidative DNA lesions. Also, all of the KD cells exhibited increased apoptosis under oxidative stress, shown by Annexin V-FITC apoptosis assays and cell cycle analysis, which indicates that NTHL1 is also required for maintaining cell viability after DNA damage. Although previous studies indicated NTHL1 can remove 8oxoG, by the in vitro BER assay, we investigated that NTHL1 involved in thymine glycol removal, but not 8oxoG. By RT-PCR, we found nthl1 and ogg1 were not stress-inducible, and nthl1 and ogg1 knock down didn’t change the expression of nei endonuclease VIII-like 1/2. In summary, NTHL1 plays important role in maintaining cell viability under oxidative stress and involves in DNA damages removal of thymine glycol, not 8oxoG.