HeLa细胞端粒酶最适反应条件及活性重建

端粒是真核细胞染色体末端的重复ＤＮＡ序列 ,其生物学功能是防止染色体ＤＮＡ降解、末端融合、非正常重组和染色体的缺失1] .由于存在“末端复制问题” ,随着老化人体细胞端粒重复序列长度不断缩短 ,但在生殖细胞中由于端粒酶的存在 ,端粒序列并不缩短 .端粒酶是由蛋白质和ＲＮＡ构成的核蛋白 ,是依赖ＲＮＡ的ＤＮＡ聚合酶 ,在ＤＮＡ3’端合成端粒重复序列2 ] .研究表明 ,在 85 %～ 95 %的人肿瘤细胞中可以检测到端粒酶的活性3 ,4 ] ,而在正常体细胞中除生殖细胞和造血干细胞等极少数细胞中存在端粒酶活性外 ,均检测不到端粒酶活性 ,这…

Optimal Catalytic Conditions of HeLa Cell Telomerase and Reconstitution of Human Telomerase Activity

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.