Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation of telomerase is often associated with immortalization of human cells and cancer. Partially purification of telomerase was obtained by chromatography of HeLa cell extract on DEAE-Sepharose FF,Spermine-Sepharose CL-6B and Sephacryl S-300. To dissect the human telomerase enzyme mechanism, the optimal catalytic conditions of telomerase were measured and the human telomerase activity was reconstituted using a purified hTR RNA. The optimum pH is from 6^5 to 8^5, the optimum temperature is from 35℃ to 40℃, the optimum Mg 2+ concentration is from 1 mmol/L to 2^5 mmol/L,the apparent binding constant (K m app) of telomerase for the primer is about 8^6 nmol/L. Partially purified human telomerase extract was treated with MNase to remove endogenous telomerase RNA(hTR),the telomerase activity was inactivation. Telomerase activity was restored by incubating MNase-treated extract with purified hTR. These results might facilitate the development of the telomerase enzyme mechanism.
端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老、癌变有着密切的关系。本实验观察了DNA依赖性蛋白激酶(DNA-dependent protein kinase,DNA-PK)抑制剂-渥曼青霉素(wortmannin,WM)对H2O2诱导的HeLa细胞端粒DNA链断裂重连接效应。结果表明WM能够显著地抑制H2O2诱导的HeLa细胞端粒DNA链断裂后的重连接作用,提示DNA-PK参与了端粒DNA链断裂损伤的修复过程。
