Regulation Of Tip60Activity And DNA Double Strand Breaks Repair Pathway By Tip60Phosphorylation And P400

DNA double strand breaks repair plays an essential role in protection of genome stability, while defect of repair pathway may leads to cancer. Therefore to uncover the mechanism of DNA double strand breaks repair is of great significance. ATM-Tip60repair pathway is the upstream of DNA double strand breaks repair pathway, the key position of the pathway. ATM kinase can activate a lot of downstream repair proteins, include p53、Chk2、 SMC1、NBS1and so on. Activation of ATM requires its acetylation by Tip60. So studies on activation of Tip60activity in the DNA double strand breaks repair pathway makes a lot of sense.As reported, the Ser86phosphorylation of Tip60activates the acetyltransferase activity of Tip60in cell autophagy, and then regulate cell autophagy pathway. Another research finds that the SANT domain of p400combines with the HAT domain of Tip60and represses the HAT activity of Tip60. This subject aims to study the regulating role of Tip60phosphorylation and p400on Tip60activity and DNA double strand breaks repair pathway. The research basically includes the effect of Tip60phosphorylation on the activity of essential proteins in the DNA double strand breaks repair ATM-Tip60pathway, cell apoptosis, interaction of Tip60with other proteins and the alteration of interaction between Tip60and p400as well as its fragment F4. We aim to clarify that how p400play negative role, and how Tip60phosphorylation play positive role in the regulation of Tip60activity and the DNA double strand breaks repair pathway.We found that mutation of Tip60phosphorylation sites leads to the diffusion of y-H2AX after the cells were irradiated, affecting the foci formation of y-H2AX.This indicated that the phosphorylation mutant makes a negative effect on the efficiency of DNA double strand breaks repair. Immunoblots demonstrated that Tip60phosphorylation has no evident impact on activity of repair proteins such as ATM, p53and so on, and there is no significant changes on the phosphorylation level of Tip60before and after DNA double strand breaks. Flow cytometry analysis indicated that double mutation of Tip60leads to an increase in cell apoptosis rate. The Co-Immunoprecipitation experiment confirmed that there was no significant variation on the interaction of Tip60and fragment F4before and after DNA double strand breaks. Immunoprecipitation experiment confirmed that mutation of Tip60 phosphorylation sites has no effect on the interaction of Tip60and other proteins.Therefore, we had a preliminary judgment that phosphorylation of Tip60at S86and S90had certain impact on the efficiency of DNA double strand breaks repair, and that the mechanism of this impact had to be determined. The regulation of p400on Tip60needed futher study to be confirmed.