The Effect Of Repair Activity Of DNA Glycosylase OGG1 On Gene Expression

Aerobic organisms always generate reactive oxygen species?ROS?resulting from endogenous aerobic metabolism and environmental exposures.When the production of ROS exceeds the removal of the intracellular antioxidants,ROS will cause oxidative damage to cellular biological macromolecules,such as protein,lipid and DNA.Damaged protein and lipid can be effectively degraded and recycled,however DNA that is genetic material need to be repaired in a timely manner.Nitrogenous base is the most vulnerable one to oxidative stress among the three structure elements composed of DNA.And guanine is most susceptible to oxidation and convert to 8-oxo-7,8-dihydroguanine?8-oxoG?due to its lower redox potential than other three bases'.8-oxoG can not only pair with cytosine,but also pair with adenine.After two cycles of continuous replication,unrepaired 8-oxoG will give a rise to G:C to T:A transversion mutations.8-oxoG DNA glycosylase OGG1 that initiates base excision repair pathway can effectively repair 8-oxoG.Recently,increasingly evidence has found that 8-oxoG not only is the most common ROS-induced adducts in the genomes,but also may serve as an epigenetic mark playing a role in regulating gene expression.More than 72%of the human gene promoter regions have a high GC content,which is easily to produce 8-oxoG under oxidative stress condition.However,our previous studies found that when exposed to high level of ROS,OGG1 itself was oxidized.Temporary inactivation OGG1 combined to 8-oxoG and recruited transcription factors such as NF-?B,formed transcription initiation complex and promoted inflammation gene transcription.In this study,we aimed to provide direct proof for our hypothesis that under oxidative stress condition,OGG1 promotes the expression of inflammatory factor independent of its BER function.To this end,we constructed OGG1 mutant with recognition activity remained but repairment activity is absent,as well as those whose recognition and repair activities are both absent.First,we have constructed GST-OGG1K249Q,GST-OGG1 K249R,GST-OGG1 C253A and GST-OGG1 D268A plasmids,and induced the expression of these fusion proteins and then purified them.Next,EMSA and cleavage assay were conducted to examine their recognition and repair activities.Furthermore,we choose OGG1 K249Q,the mutant lacking of repair activity but still able to bind with 8-oxoG,and OGG1 D268A which completely lose its recognition and repair activities for 8-oxoG to study transcription function of OGG1 in vivo.We constructed YFP-OGG1 K249Q and YFP-OGG1 D268A plasmids and transfected them into MEF Ogg1^-/-cells.After adding proinflammatory stimulus TNF-?for different time course,we tested Cxcl2 mRNA expression level by real-time PCR.The results showed that under oxidative stress OGG1 promotes the expression of inflammatory factor,which does not depend on its repair of 8-oxoG.This is a novel regulation mechanism of gene expression for BER enzyme OGG1.We promise this study would provide theoretical basis for the research of OGG1 inhibitors,even has a certain referential value for related diseases targeted therapies.