Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA.
Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA.
Nature. 2022 Aug;608(7924):813-818. doi: 10.1038/s41586-022-04931-7. Epub 2022 Jul 13.
Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand and TPP1 (in complex with TIN2) recruiting telomerase via interaction with telomerase reverse transcriptase (TERT). The telomere DNA ends are replicated and maintained by telomerase, for the G-strand, and subsequently DNA polymerase α-primase (PolαPrim), for the C-strand. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN1 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.
端粒是线性染色体的物理末端。它们由双链 DNA 组成，其中包含短的重复序列（例如四膜虫热变形体中的 G 链上的 TTGGGG），G 链上有单链 3' 突出，人类中有六种庇护蛋白：TPP1、POT1、TRF1、TRF2、RAP1 和 TIN2。TPP1 和 POT1 与 3' 突出结合，POT1 结合 G 链，TPP1（与 TIN2 复合物）通过与端粒酶逆转录酶（TERT）的相互作用招募端粒酶。端粒 DNA 末端由端粒酶复制和维持，用于 G 链，随后由 DNA 聚合酶 α-引发酶（PolαPrim）用于 C 链。PolαPrim 活性由异三聚体复合物 CTC1-STN1-TEN1（CST）刺激，但 PolαPrim 和 CST 招募到端粒末端的结构基础仍不清楚。在这里，我们报告了在端粒酶全酶的背景下，四膜虫 CST 在没有和存在 PolαPrim 以及单独的 PolαPrim 时的低温电子显微镜（cryo-EM）结构。四膜虫 Ctc1 通过 cryo-EM 和 NMR 光谱揭示的柔性 Ctc1 结合基序结合端粒酶亚基 p50，即 TPP1 同源物。PolαPrim 聚合酶亚基 POLA1 结合 Ctc1 和 Stn1，其与 Ctc1 的界面形成 G 链 DNA 进入 POLA1 活性位点的入口。因此，我们提供了一个快照，其中包含四个关键成分，它们在单个活性复合物-端粒酶核心核糖核蛋白、p50、CST 和 PolαPrim 中共同作用，用于端粒 DNA 合成，为 CST 和 PolαPrim 的招募以及 G 链和 C 链合成之间的交接提供了见解。
