Interaction hub critical for telomerase recruitment and primer-template handling for catalysis

Telomerase processively adds telomeric DNA repeats to chromosome ends using catalytic protein subunit TERT and a template on its RNA subunit TR. Mammalian telomerase is recruited to telomeres by the TEL patch and NOB regions of shelterin component TPP1. Recent cryo-EM structures of human telomerase reveal that a composite TERT TEN-(IFD-TRAP) domain interacts with TPP1. Here, we generate TERT mutants to demonstrate that a three-way TEN-(IFD-TRAP)-TPP1 interaction is critical for telomerase recruitment to telomeres and processive telomere repeat addition. Single mutations of IFD-TRAP at its interface with TR or the DNA primer impair telomerase catalysis. We further reveal the importance of TERT motif 3N and TEN domain loop ⁹⁹FGF¹⁰¹ in telomerase action. Finally, we demonstrate that TPP1 TEL patch loop residue F172, which undergoes a structural rearrangement to bind telomerase, contributes to the human-mouse species specificity of the telomerase-TPP1 interaction. Our study provides insights into the multiple functions of TERT IFD-TRAP, reveals novel TERT and TPP1 elements critical for function, and helps explain how TPP1 binding licenses robust telomerase action at natural chromosome ends.