The Preliminary Study About The Function Of Purα With Its Related Protein Complex Involved In DNA Damage Repair

One of the major problems in cancer therapy is the occurrence of chemoresistance. Amongthe classic antineoplastic compounds, it has been shown that a high effcacy of certain forms ofDNA repair promotes genomic rearrangements when the level of DSB is high. Therefore, furtherstudies about the DNA repair machinery is important for much better understanding of the DNAdamage response of tumour cells.Purα has a high affinity for single-stranded oligonucleotides, DNA or RNA. With regard totumorigenesis, many studies have indicated that Purα has the properties of a tumor suppressorprotein through its interactions with pRb, E2F1, etc. In addition, although some reports show thatPurα may be involved in DNA repair, the mechanism of its function is not clear. In our previousstudies, By using TAP experiment and Mass spectrometry, we analyzed the components of Puraprotein complex, which is include the Ku70and Ku80, which play important roles in DNArepairis cleared. Therefore, the demonstration of a physical association between Pura and Ku70/80hasprovided a useful insight into the study of repair mechanism. We can do in-depth on Purα proteinthat is involved at DNArepair and the cellular response to DNAreplication stress.In this work, the immunoprecipitation experiment proved that Purα protein interacts withKu70/80proteins. Besides we build pGADT7-KU80, pGBKT7-KU80, pGADT7-KU70andpGBKT7-KU70plasmids, we also successfully transformed the plasmid into yeast cells, inpreparation for subsequent mating experiments. The using of MTT assay showed that the role ofPurα in the cellular response to three anticancer drugs using that we generated293T cells whichcan express FLAG tag Purα fusion protein. We found that Purα expressed cells are lesshypersensitive to doxorubicin and the methyl methanesulfonate. The experiments ofImmunocytochemistry showed that with the using doxorubicin it is showed a phenomenon of theco-localization between Purα and Ku70/80protein in the nucleus. Purα is mainly located in thecytoplasm, after the treatment with doxorubicin, Purα translocated to the nucleus and theexpression of Purα and Ku70/80proteins were obviously higher, suggest a role for Purα innuclear processes triggered by doxorubicin. These results suggest a role for Purα is involved inthe repair of DSBs induced by doxorubicin. In addition, the success of constructin with low andstable protein expression Purα Hela and U2OS cell lines, which can be the foundation for furtherexperiments.