Characterization of t-loop formation by TRF2.
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ABSTRACT:
SUBMITTER: Timashev LA
PROVIDER: S-EPMC7529409 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We have applied a super-resolution fluorescence imaging method, stochastic optical reconstruction microscopy (STORM), to visualize the structure of functional telomeres and telomeres rendered dysfunctional through removal of shelterin proteins. The STORM images showed that functional telomeres frequently exhibit a t-loop configuration. Conditional deletion of individual components of shelterin showed that TRF2 was required for the formation and/or maintenance of t-loops, whereas deletion of TRF1, Rap1, or the POT1 proteins (POT1a and POT1b) had no effect on the frequency of t-loop occurrence. Within the shelterin complex, TRF2 uniquely serves to protect telomeres from two pathways that are initiated on free DNA ends: classical nonhomologous end-joining (NHEJ) and ATM-dependent DNA damage signaling. The TRF2-dependent remodeling of telomeres into t-loop structures, which sequester the ends of chromosomes, can explain why NHEJ and the ATM signaling pathway are repressed when TRF2 is present.
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Project description:A growing body of literature suggests that the homologous recombination/repair (HR) pathway cooperates with components of the shelterin complex to promote both telomere maintenance and nontelomeric HR. This may be due to the ability of both HR and shelterin proteins to promote strand invasion, wherein a single-stranded DNA (ssDNA) substrate base pairs with a homologous double-stranded DNA (dsDNA) template displacing a loop of ssDNA (D-loop). Rad51 recombinase catalyzes D-loop formation during HR, and telomere repeat binding factor 2 (TRF2) catalyzes the formation of a telomeric D-loop that stabilizes a looped structure in telomeric DNA (t-loop) that may facilitate telomere protection. We have characterized this functional interaction in vitro using a fluorescent D-loop assay measuring the incorporation of Cy3-labeled 90-nucleotide telomeric and nontelomeric substrates into telomeric and nontelomeric plasmid templates. We report that preincubation of a telomeric template with TRF2 inhibits the ability of Rad51 to promote telomeric D-loop formation upon preincubation with a telomeric substrate. This suggests Rad51 does not facilitate t-loop formation and suggests a mechanism whereby TRF2 can inhibit HR at telomeres. We also report a TRF2 mutant lacking the dsDNA binding domain promotes Rad51-mediated nontelomeric D-loop formation, possibly explaining how TRF2 promotes nontelomeric HR. Finally, we report telomere repeat binding factor 1 (TRF1) promotes Rad51-mediated telomeric D-loop formation, which may facilitate HR-mediated replication fork restart and explain why TRF1 is required for efficient telomere replication.
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Project description:The protection of telomere ends 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 accurate 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 the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.
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Project description:The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to prevent catastrophic t-loop processing by structure-specific nucleases. We show that the TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1(R1264H). Conversely, we define a TRF2(I124D) substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1(R1264H) mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase.
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Project description:Telomere-repeat-encoding RNA (referred to as TERRA) has been identified as a potential component of yeast and mammalian telomeres. We show here that TERRA RNA interacts with several telomere-associated proteins, including telomere repeat factors 1 (TRF1) and 2 (TRF2), subunits of the origin recognition complex (ORC), heterochromatin protein 1 (HP1), histone H3 trimethyl K9 (H3 K9me3), and members of the DNA-damage-sensing pathway. siRNA depletion of TERRA caused an increase in telomere dysfunction-induced foci, aberrations in metaphase telomeres, and a loss of histone H3 K9me3 and ORC at telomere repeat DNA. Previous studies found that TRF2 amino-terminal GAR domain recruited ORC to telomeres. We now show that TERRA RNA can interact directly with the TRF2 GAR and ORC1 to form a stable ternary complex. We conclude that TERRA facilitates TRF2 interaction with ORC and plays a central role in telomere structural maintenance and heterochromatin formation.
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