Study On The Mechanism Of DNA Polymerase ? (Pol?) Interaction Protein In Base Excision Repair

The human genome is constantly exposed to a variety of endogenous and exogenous DNA damaging agents that likely to induce DNA damage.This DNA damage,if not repaired,could affect DNA replication and transcription as well as chromosomal structure,and even cause the body to produce a variety of diseases such as cancer.To repair DNA damage,the human has evolved multiple DNA damage and repair systems to repair various damages.Base excision repair(BER)as one of the important DNA damage repair systems plays an important role in repairing DNA damage caused by alkylation,oxidation,AP sites and single-strand breaks.In order to ensure that base excision repair in a timely,accurate and efficient manner to repair damaged DNA,there are various mechanisms for its regulation,including protein and protein interactions and post-translational modifications and so on.In this study,we focused on the core protein of BER,DNA Polymerase beta(Pol ?),seeking out new Pol ? interaction proteins and studying the effects on its function and BER involved.To this end,by mass spectrometry and co-immunoprecipitation technologies,we identified that glyceraldehyde-3-phosphate dehydrogenase(GAPDH)and Histone deacetylase 3(HDAC3)are novel Pol ? interacting proteins and mainly investigated the mechanisms of their role on BER pathway.1.GAPDH binds with Pol ? and participates in base excision repair(1)Demonstrate a direct binding of GAPDH and Pol ? by a series of co-immunoprecipitation experiments,and founding that DNA damage stimulation can enhance their binding ability(2)Study the effects of the bindings between GAPDH and Pol ? on their stability and activity.The results showed that the binding of GAPDH and Pol ? was not associated with their stability but with their activity.On the aspect of activity,GAPDH promotes Pol ? polymerase activity while Pol ? does not affect GAPDH glycolysis activity.(3)Since Pol ? activity is closely associated with the efficiency of its involved BER,we further investigated the affects of GAPDH on the BER efficiency.To this end,we used a purified recombinant protein or cell lysate to reconstitute the BER pathway.The results showed that GAPDH significantly promotes BER efficiency.(4)We studied the mechanism of GAPDH nuclear transport involved in DNA base excision repair under DNA damage.We found that DNA damage treatments promote the activation of Src signaling,and activate Src mediatting phosphorylation of GAPDH promoting nuclear translocation.Using of small interfering RNA to knockdown GAPDH expression or to inhibit its nuclear translocation by Src inhibitors makes cells more sensitive to DNA damage agents.(5)Targeting DNA damage pathways are often used as a tumor therapy and targeting proteins in BER pathways for killing cancer cells have also been extensively studied.Based on the important functions of GAPDH in the BER pathway,we initially explored the role of knockdown of GAPDH in tumor chemotherapy.Xenograft models of colon cancer cells showed that specifically inhibiting the expression of GAPDH in the tumor increases its sensitivity to the chemotherapy drug 5-FU,suggesting that GAPDH may be a new target in combination with tumor chemotherapy.2.HDAC3 binds with Pol ? and participates in BER(1)HDAC3 can directly bind with Pol ? in cells.By constructing different truncations of HDAC3 and combining with immunoprecipitation experiments,we found that the N-terminal of 1-180 amino acids within HDAC3 is a necessary segment for its binding to Pol ?.(2)DNA damage and stimulation can enhance the interaction of HDAC3 and Pol ? in cells.(3)HDAC3 can promote the polymerase activity of Pol ? and enhance the efficiency of BER.(4)Inhibition of HDAC3 expression in cells reduces the efficiency of intracellular BER,resulting in a more accumulation of intracellular DNA damage and increasing the sensitivity of cells to DNA damage agents.Taken together,focusing on the central position of Pol ? in the BER pathway,we have identified its new interacting proteins including GAPDH and HDAC3.We have demonstrated their effects on Pol ? function and BER process.Our research have further enriched the binding proteins of Pol ? and expanded the mechanisms of glyceraldehyde-3-phosphate dehydrogenase and histone deacetylase in DNA damage repair.