Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10065, USA.
Nucleic Acids Res. 2018 Apr 6;46(6):3088-3102. doi: 10.1093/nar/gky117.
Telomerase is a specialized enzyme that maintains telomere length by adding DNA repeats to chromosome ends. The catalytic protein subunit of telomerase utilizes the integral telomerase RNA to direct telomere DNA synthesis. The telomerase essential N-terminal (TEN) domain is required for enzyme function; however, the precise mechanism of the TEN domain during catalysis is not known. We report a single-molecule study of dynamic TEN-induced conformational changes in its nucleic acid substrates. The TEN domain from the yeast Candida parapsilosis (Cp) exhibits a strong binding preference for double-stranded nucleic acids, with particularly high affinity for an RNA-DNA hybrid mimicking the template-product complex. Surprisingly, the telomere DNA repeat sequence from C. parapsilosis forms a DNA hairpin that also binds CpTEN with high affinity. Mutations to several residues in a putative nucleic acid-binding patch of CpTEN significantly reduced its affinity to the RNA-DNA hybrid and telomere DNA hairpin. Substitution of comparable residues in the related Candida albicans TEN domain caused telomere maintenance defects in vivo and decreased primer extension activity in vitro. Collectively, our results support a working model in which dynamic interactions with telomere DNA and the template-product hybrid underlie the functional requirement for the TEN domain during the telomerase catalytic cycle.
端粒酶是一种专门的酶，通过在染色体末端添加 DNA 重复序列来维持端粒长度。端粒酶的催化蛋白亚基利用完整的端粒酶 RNA 来指导端粒 DNA 的合成。端粒酶必需的 N 端（TEN）结构域是酶功能所必需的；然而，在催化过程中 TEN 结构域的确切机制尚不清楚。我们报告了一项关于其核酸底物中 TEN 诱导的构象变化的单分子研究。来自酵母近平滑假丝酵母（Cp）的 TEN 结构域对双链核酸表现出强烈的结合偏好，对模拟模板-产物复合物的 RNA-DNA 杂交体具有特别高的亲和力。令人惊讶的是，来自 C. parapsilosis 的端粒 DNA 重复序列形成一个 DNA 发夹，也能与 CpTEN 高亲和力结合。CpTEN 中一个假定的核酸结合点的几个残基发生突变，显著降低了其与 RNA-DNA 杂交体和端粒 DNA 发夹的亲和力。在相关的白色念珠菌 TEN 结构域中取代类似的残基，会导致体内端粒维持缺陷和体外引物延伸活性降低。总之，我们的结果支持这样一个工作模型，即在端粒酶催化循环中，TEN 结构域与端粒 DNA 和模板-产物杂交体的动态相互作用是其功能所必需的。
