The Regulatory Effects Of Pur? On PARP1 Gene Expression And Their Collaborative Roles In DNA Damage And Repair

Objective:Pur? is a multifunctional protein, it plays an important role in multiple link,especially in the developmental processes of the nervous system. The role of Pur? in damaged DNA repair is very obvious. Studies have also shown that Pur? plays the role of supervision in the DSBs repair which is caused by the stall of replication fork, which is helpful to maintain the stability of the genomic DNA, the expression of Pur? has an important clinical significance in the pathogenesis of neurodegenerative diseases. The expression of Pur? is specific, and it is closely related to the development of neurons and synaptogenesis. Poly(ADP-ribose) polymerase 1(PARP1) is a ribozyme in eukaryote cells that catalyzes the process of poly(ADP-ribosyl) ation. PARP1 is a highly conserved DNA binding protein that can identify a very low level of DNA damage, activate the DNA damage repair system, repair damaged DNA; it will start the apoptosis when the DNA damage is very serious and unable to repair in order to maintain the genetic integrity. Our previous work demonstrated that Pur?and PARP1 could bind together in vitro. However, whether Pur? could regulate the PARP1 gene expression or not, and how does Pur? perform the regulatory function, is that regulation a positive or a negative, where the functional domain of Pur? located, and all of these are still not clear. In this research, we investigate the regulatory effects of Pur? on PARP1 gene expression and the regulatory mechanisms of Pur? on PARP1. We investigate the regulatory effects of the four deletions of Pura on PARP1 gene expression, in order to indentify the active center of Pura. We also apply the four deletions of Pura on APP gene expression; the purpose is to further confirm the active center of Pura.Method:(1) Primers were designed according to the Pur? gene sequence. We construct p EGFP-Pur? mutants(deletions) plasmids(p EGFP-P1?p EGFP-P2?p EGFP-P3?p EGFP-P4).(2) The pull-down assay and Co-Immunoprecipitation are employed to detect the physical interaction between Pur? and PARP1 proteins.(3) Using luciferase assay, Real time PCR and Western blotting assay to verify the function of the full length and different deletions of Pur?protein on PARP1 gene expression in HT22 cells.(4) The 2m M of HU was applied to establish the DNA damage model in HT22 cells, using immunofluorescence assay to ascertain the intracellular distribution of Pur? and PARP1 proteins. At the same time, using Western blotting assay to discuss the functions of overexpression Pur? protein on the PARP1 in DNA damage.(5) Using Western blotting assay to explore the functions of overexpression Pur?protein on the signal molecule of m TOR in the HT22 cells, in order to identify the regulatory mechanisms of Pur? on PARP1 gene expression.Results:(1) The results of double-endonuclease digestions and sequencing analysis proved that recombinant plasmid p EGFP-Pur? and their deleted mutants' plasmids(p EGFP-P1,p EGFP-P2, p EGFP-P3 and p EGFP-P4) were successfully constructed.(2) The results of Pull-down assay and Co-Immunoprecipitation demonstrated the physical interaction between the Pur? and PARP1 proteins.(3) The results of Luciferase assay, Real time PCR and Western blotting assay confirmed the full-length of Pur? has largest upregulatory effect on the PARP1 gene expression; in the four deletions, P3 can increase the PARP1 and APP gene expression levels;(4) The results of Immunofluorescence assay indicate that Pur? and PARP1 proteins co-localized inside the nucleus in the status of DNA damage. At the meantime, the result of Western blotting assay indicates Pur? could promote PARP1 to play its biological function.(5) The result of Western blotting assay indicates Pur? regulates PARP1 gene expression by m TOR pathway.Conclusion:(1) Pur? protein and PARP1 protein have physical interactions.(2) Pur? can upregulate the expression of PARP1 gene expression.(3) To keep the intact structure in N-terminal of the Pur? may be essential for maintenance of the functions of Pur?.(4) Pur?protein and PARP1 protein will flock to the nucleus and cooperates to repair the damage DNA. At the meantime, Pur? could promote PARP1 to play its biological functions.(4) Pur? regulates PARP1 gene expression by m TOR pathway.