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Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo

Abstract

Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPα and TEBPβ. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPα in attaching telomeres in the nucleus and in recruiting TEBPβ to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPβ in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle–dependent phosphorylation of TEBPβ.

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Figure 1: Schematic representation of a macronuclear nanochromosome.
Figure 2: TEBPα attaches telomeres to a subnuclear structure and recruits TEBPβ to the attachment sites.
Figure 3: Silencing of TEBPα and TEBPβ gene expression by RNAi.
Figure 4: Folding of telomeric G-overhangs into an antiparallel G-quadruplex DNA structure requires both TEBPα and TEBPβ in vivo.
Figure 5: TEBPβ requires TEBPα in order to interact with telomeric DNA in vitro.
Figure 6: Both TEBPs are required for G-quadruplex DNA formation and the basic C terminus of TEBPβ is essential.
Figure 7: Phosphorylated TEBPβ does not interact with telomeric DNA bound by TEBPα in vitro.
Figure 8: TEBPβ is phosphorylated during S phase, and inhibition of phosphorylation prevents the G-quadruplex DNA structure from being resolved during replication.

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Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to H.J.L., a European Molecular Biology Organization short-term fellowship to K.P. and a Wenner-Gren Foundations fellowship to T.S. We thank T. Cech (University of Colorado, Boulder, Colorado, USA) for providing antibodies against the TEBP subunits and C. Schaffitzel (Swiss Federal Institute of Technology, Zurich) for providing antibodies against G-quadruplex DNA structures.

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Correspondence to Daniela Rhodes or Hans Joachim Lipps.

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Supplementary information

Supplementary Fig. 1

A ClustalW sequence alignment of TEBPβ from Stylonychia lemnae and Oxytricha nova. (PDF 109 kb)

Supplementary Table 1

Stability of telomeric DNA upon RNAi mediated silencing of TEBPα or TEBPβ expression: no RNAi. (PDF 16 kb)

Supplementary Table 2

Stability of telomeric DNA upon RNAi mediated silencing of TEBPa or TEBPb expression: TEBPα RNAi. (PDF 16 kb)

Supplementary Table 3

Stability of telomeric DNA upon RNAi mediated silencing of TEBPa or TEBPb expression: TEBPβ RNAi. (PDF 16 kb)

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Paeschke, K., Simonsson, T., Postberg, J. et al. Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nat Struct Mol Biol 12, 847–854 (2005). https://doi.org/10.1038/nsmb982

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