Multiple Mechanisms for Elongation Processivity within the Reconstituted Tetrahymena Telomerase Holoenzyme
Multiple Mechanisms for Elongation Processivity within the Reconstituted Tetrahymena Telomerase Holoenzyme
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DOI：
10.1074/jbc.m110.119172
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发表时间：
2010-05-28
影响因子：
4.8
通讯作者：
Collins, Kathleen
中科院分区：
文献类型：
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作者：
Min, Bosun;Collins, Kathleen
为了维持端粒，端粒酶进化出一种独特的生物化学活性：利用单链RNA模板合成单链DNA重复序列。四膜虫端粒酶全酶的高重复添加持续合成能力（RAP）需要催化核心与端粒衔接子复合物（TASC）和RPA 1相关亚基（p82或Teb 1）的结合。在这里，我们使用DNA结合和全酶重建测定来研究Teb 1和TASC赋予高RAP的机制。我们发现，TASC协会与重组端粒酶催化核心增加酶的活性。Teb 1 C-末端结构域与TASC的后续关联赋予了高RAP的能力，即使Teb 1 C-末端结构域不提供高亲和力DNA相互作用位点。有效的RAP还需要抑制由中心Teb 1 DNA结合结构域（DBD）介导的新生产物折叠。Teb 1的这些序列特异性高亲和力DBD可以在功能上被四膜虫Rpa 1的类似DBD取代，以抑制新生产物折叠，但前提是Rpa 1高亲和力DBD通过Teb 1 C-末端结构域物理地束缚在全酶环境中。总的来说，我们的研究结果揭示了蛋白质-DNA和蛋白质-蛋白质相互作用的多种机制和多种表面，这些机制和表面在单链核酸产物的合成中引起延伸持续合成能力。
To maintain telomeres, telomerase evolved a unique biochemical activity: the use of a single-stranded RNA template for the synthesis of single-stranded DNA repeats. High repeat addition processivity (RAP) of the Tetrahymena telomerase holoenzyme requires association of the catalytic core with the telomere adaptor subcomplex (TASC) and an RPA1-related subunit (p82 or Teb1). Here, we used DNA binding and holoenzyme reconstitution assays to investigate the mechanism by which Teb1 and TASC confer high RAP. We show that TASC association with the recombinant telomerase catalytic core increases enzyme activity. Subsequent association of the Teb1 C-terminal domain with TASC confers the capacity for high RAP even though the Teb1 C-terminal domain does not provide a high-affinity DNA interaction site. Efficient RAP also requires suppression of nascent product folding mediated by the central Teb1 DNA-binding domains (DBDs). These sequence-specific high-affinity DBDs of Teb1 can be functionally substituted by the analogous DBDsof Tetrahymena Rpa1 to suppress nascent product folding but only if the Rpa1 high-affinity DBDs are physically tethered into holoenzyme context though the Teb1 C-terminal domain. Overall, our findings reveal multiple mechanisms and multiple surfaces of protein-DNA and protein-protein interaction that give rise to elongation processivity in the synthesis of a single-stranded nucleic acid product.