CDK phosphorylation of TRF2 controls t-loop dynamics during the cell cycle.

Grzegorz Sarek,Ambrosius P Snijders,Helen R Flynn,Valerie Borel,David Van Ly,Xiao-Feng Zheng,Panagiotis Kotsantis,Pol Margalef,Anthony J Cesare,Simon J Boulton,Phil Ruis,Dipanjan Chowdhury

doi:10.1038/s41586-019-1744-8

Grzegorz Sarek, Ambrosius P Snijders + Show 10 more

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https://doi.org/10.1038/s41586-019-1744-8

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Telomere end-protection by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3’ single-stranded telomere end can assemble into a lasso-like t-loop configuration1,2, which has been proposed to safeguard chromosome ends from being recognized as DNA double strand breaks2. Mechanisms must also exist to transiently disassemble t-loops to allow faithful telomere replication and to permit telomerase access to the 3’-end to solve the end replication problem. However, the regulation and physiological importance of t-loops in end-protection remains uncertain. Here, we identify a CDK phosphorylation site in the shelterin subunit, TRF2 (Ser365), whose dephosphorylation in S-phase by the PP6C/R3 phosphatase provides a narrow window during which the helicase RTEL1 is able to transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 on Ser365 outside of S-phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate ATM activation, but also counteracts replication conflicts at DNA secondary structures arising within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.

Highlights

We identify a CDK phosphorylation site in the shelterin subunit, TRF2 (Ser365), whose dephosphorylation in S-phase by the PP6C/R3 phosphatase provides a narrow window during which the helicase RTEL1 is able to transiently access and unwind t-loops to facilitate telomere replication
Telomere homeostasis is critically dependent on the function of the shelterin complex but how this is regulated during the cell cycle remains uncertain
Through phospho-proteomic analysis of the shelterin complex, we identified a putative CDK2 phosphorylation site in human TRF2 at Ser[365] (Ser[367] in mouse; Extended Data Fig. 1a), which is abolished by λPPase treatment (Fig. 1a, left and middle panels) or mutation of the phospho-site to alanine (Myc-TRF2S367A; Fig. 1a, right panel)

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Introduction

TRF2F/- mouse embryonic fibroblasts (MEFs) complemented with either wt or phospho-dead (Myc-TRF2S367A) or phospho-mimetic (Myc-TRF2S367D and Myc-TRF2S367E) mutants lacked telomere fusions (Fig. 1c; left panel; Extended Data Fig. 1c, d). The TRF2Ser[367] mutants retained interactions with other shelterin proteins, including TRF1 and Rap[1], and depletion of Rap[1] did not result in telomere fusions in cells expressing the Myc-TRF2S367A mutant (4 and Extended Data Fig. 2a-c).

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