Dataset Information

Telomere Recognition and Assembly Mechanism of Mammalian Shelterin.

ABSTRACT: Shelterin is a six-subunit protein complex that plays crucial roles in telomere length regulation, protection, and maintenance. Although several shelterin subunits have been studied in vitro, the biochemical properties of the fully assembled shelterin complex are not well defined. Here, we characterize shelterin using ensemble biochemical methods, electron microscopy, and single-molecule imaging to determine how shelterin recognizes and assembles onto telomeric repeats. We show that shelterin complexes can exist in solution and primarily locate telomeric DNA through a three-dimensional diffusive search. Shelterin can diffuse along non-telomeric DNA but is impeded by nucleosomes, arguing against extensive one-dimensional diffusion as a viable assembly mechanism. Our work supports a model in which individual shelterin complexes rapidly bind to telomeric repeats as independent functional units, which do not alter the DNA-binding mode of neighboring complexes but, rather, occupy telomeric DNA in a "beads on a string" configuration.

SUBMITTER: Erdel F

PROVIDER: S-EPMC5225662 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Telomere Recognition and Assembly Mechanism of Mammalian Shelterin.

Erdel Fabian F Kratz Katja K Willcox Smaranda S Griffith Jack D JD Greene Eric C EC de Lange Titia T

Cell reports 20170101 1

Shelterin is a six-subunit protein complex that plays crucial roles in telomere length regulation, protection, and maintenance. Although several shelterin subunits have been studied in vitro, the biochemical properties of the fully assembled shelterin complex are not well defined. Here, we characterize shelterin using ensemble biochemical methods, electron microscopy, and single-molecule imaging to determine how shelterin recognizes and assembles onto telomeric repeats. We show that shelterin co ...[more]

PMID: 28052260

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Project description:Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (human AKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factor identified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere dysfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication.

Dataset Information

Telomere Recognition and Assembly Mechanism of Mammalian Shelterin.

Publications

Telomere Recognition and Assembly Mechanism of Mammalian Shelterin.

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