Telomeres are the physical ends of linear chromosomes, composed of short repeating sequences (e.g. TTGGGG in Tetrahymena for the G-strand) of double-stranded DNA with a single-strand 3'-overhang of the G-strand and a group of proteins called shelterin. Among these, TPP1 and POT1 associate with the 3'-overhang, with POT1 binding the G-strand and TPP1 recruiting telomerase via interaction with telomerase reverse transcriptase (TERT). The ends of the telomeric DNA are replicated and maintained by telomerase, for the G-strand, and subsequently DNA Polymerase α-Primase (PolαPrim), for the C-strand. PolαPrim is stimulated by CTC1-STN1-TEN1 (CST), but the structural basis of both PolαPrim and CST recruitment to telomere ends remains unknown. Here we report cryo-EM structures of Tetrahymena CST in the context of telomerase holoenzyme, both in the absence and presence of PolαPrim, as well as of PolαPrim alone. Ctc1 binds telomerase subunit p50, a TPP1 ortholog, on a flexible Ctc1 binding motif unveiled jointly by cryo-EM and NMR spectroscopy. PolαPrim subunits are arranged in a catalytically competent conformation, in contrast to previously reported autoinhibited conformation. Polymerase POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. Together, we obtained a snapshot of four key players required for telomeric DNA synthesis in a single complex-telomerase core RNP, p50/TPP1, CST and PolαPrim-that provides unprecedented insights into CST and PolαPrim recruitment and handoff between G-strand and C-strand synthesis.
Structural basis of sodium–potassium exchange of a human telomeric DNA quadruplex without topological conversion
