Coordinated DNA dynamics during the human telomerase catalytic cycle.

The human telomerase reverse transcriptase (hTERT) utilizes a template within the integral RNA subunit (hTR) to direct extension of telomeres. Telomerase exhibits repeat addition processivity (RAP) and must therefore translocate the nascent DNA product into a new RNA:DNA hybrid register to prime each round of telomere repeat synthesis. Here we use single-molecule FRET and nuclease protection assays to monitor telomere DNA structure and dynamics during the telomerase catalytic cycle. DNA translocation during RAP proceeds through a previously uncharacterized kinetic sub-step during which the 3′-end of the DNA substrate base pairs downstream within the hTR template. The rate constant for DNA primer re-alignment reveals this step is not rate-limiting for RAP, suggesting a second slow conformational change repositions the RNA:DNA hybrid into the telomerase active site and drives the extrusion of the 5′-end of the DNA primer out of the enzyme complex.

人端粒酶逆转录酶（hTERT）利用其组成性RNA亚基（hTR）内的一个模板来指导端粒的延伸。端粒酶表现出重复添加持续性（RAP），因此必须将新生的DNA产物转移到一个新的RNA：DNA杂交位点，以便启动每一轮端粒重复序列的合成。在此，我们利用单分子荧光共振能量转移（FRET）和核酸酶保护分析来监测端粒酶催化循环过程中端粒DNA的结构和动态。在RAP过程中的DNA转移通过一个之前未被描述的动力学亚步骤进行，在此步骤中，DNA底物的3′端在hTR模板内与下游的碱基配对。DNA引物重新排列的速率常数表明，这一步骤对于RAP不是限速步骤，这意味着第二个缓慢的构象变化将RNA：DNA杂交体重新定位到端粒酶活性位点，并促使DNA引物的5′端从酶复合物中挤出。