Structural Insights into Yeast Telomerase Recruitment to Telomeres

Abstract

Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to telomeres occurs through its Ku and Est1 subunits via independent interactions with telomerase RNA (TLC1) and telomeric proteins Sir4 and Cdc13, respectively. However, the structures of the molecules comprising these telomerase-recruiting pathways remain unknown. Here, we report crystal structures of the Ku heterodimer and Est1 complexed with their key binding partners. Two major findings are as follows: (1) Ku specifically binds to telomerase RNA in a distinct, yet related, manner to how it binds DNA; and (2) Est1 employs two separate pockets to bind distinct motifs of Cdc13. The N-terminal Cdc13-binding site of Est1 cooperates with the TLC1-Ku-Sir4 pathway for telomerase recruitment, whereas the C-terminal interface is dispensable for binding Est1 in vitro yet is nevertheless essential for telomere maintenance in vivo. Overall, our results integrate previous models and provide fundamentally valuable structural information regarding telomere biology.

The Sir4 H‐ BRCT domain interacts with phospho‐proteins to sequester and repress yeast heterochromatin

“…Sir4 binds both Sir2 and Sir3, and therefore is thought to provide a scaffold for SIR complex formation (Hecht et al, 1996;Moazed et al, 1997;Rudner et al, 2005). The Ku-binding motif (KBM, residues 104-115; green box in Fig 1A) of Sir4 is involved in recruiting telomerase to telomeres through interaction between telomere-bound Sir4 and the Ku-telomerase complex (Schober et al, 2009;Chen et al, 2018). The Ku-binding motif (KBM, residues 104-115; green box in Fig 1A) of Sir4 is involved in recruiting telomerase to telomeres through interaction between telomere-bound Sir4 and the Ku-telomerase complex (Schober et al, 2009;Chen et al, 2018).…”

“…Sir4 comprises several well-studied domains (Figs 1A and EV1). The Ku-binding motif (KBM, residues 104-115; green box in Fig 1A) of Sir4 is involved in recruiting telomerase to telomeres through interaction between telomere-bound Sir4 and the Ku-telomerase complex (Schober et al, 2009;Chen et al, 2018). yKu80/ yKu70 interacts with Sir4 regions 1-270 and 747-1,358 to recruit the SIR complex to telomeres and facilitates anchoring of silenced chromatin at the nuclear periphery (Tsukamoto et al, 1997;Laroche et al, 1998;Hediger et al, 2002;Luo et al, 2002;Roy et al, 2004;Schober et al, 2009).…”

Proteomic Analysis of DNA Synthesis on a Structured DNA Template in Human Cellular Extracts: Interplay Between NHEJ and Replication‐Associated Proteins

“…[ 26 ] In addition, Ku associates to a specific stem‐loop structure of the telomerase RNA in both yeast [ 27 ] and human [ 28 ] and this interaction is crucial for recruiting the active reverse transcriptase complex. [ 29 ]…”

Loss of Ku’s DNA end binding activity affects telomere length via destabilizing telomere-bound Est1 rather than altering TLC1 homeostasis

“…Additional subunits of telomerase include Est1, which binds a stem-loop structure of TLC1 11 , and Est3, which associates with the telomerase complex via binding sites on Est1 and Est2 12,13 . Est1 is involved in the recruitment of telomerase to the telomere during late S phase through its association with Cdc13, a single-stranded DNA binding protein that binds the terminal 3' overhang of the telomeric G-rich strand with high affinity [14][15][16][17][18][19] . In addition, there is evidence to support a second role for Est1 subsequent to telomerase recruitment 19,20 , which is often referred to as activation 14 .…”

“…Sir4 localizes to telomeres via its association with 15-20 Rap1 molecules that are bound directly along the ~300 basepairs (bp) of telomeric DNA 36,37 . Sir4 also binds Ku and this interaction is an important pathway for telomerase recruitment to the telomere 14,38 . In support of this, Est2 enrichment at telomere VI-R is greatly reduced in strains bearing mutations in key Ku80 binding residues on Sir4 14 .…”