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Yeast telomerase subunit Est1p has guanine quadruplex–promoting activity that is required for telomere elongation

Abstract

Telomeres are eukaryotic protein–DNA complexes found at the ends of linear chromosomes that are essential for maintaining genome integrity and are implicated in cellular aging and cancer. The guanine (G)-rich strand of telomeric DNA, usually elongated by the telomerase reverse transcriptase, can form a higher-order structure known as a G-quadruplex in vitro and in vivo. Several factors that promote or resolve G-quadruplexes have been identified, but the functional importance of these structures for telomere maintenance is not well understood. Here we show that the yeast telomerase subunit Est1p, known to be involved in telomerase recruitment to telomeres, can convert single-stranded telomeric G-rich DNA into a G-quadruplex structure in vitro in a Mg2+-dependent manner. Cells carrying Est1p mutants deficient in G-quadruplex formation in vitro showed gradual telomere shortening and cellular senescence, indicating a positive regulatory role for G-quadruplex in the maintenance of telomere length.

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Figure 1: Est1p promotes yeast telomeric single-stranded G-rich DNA to form G-quadruplex.
Figure 2: Substrate requirement for the G-quadruplex–promoting activity of GSTEst1p.
Figure 3: The G-quadruplex formation promoted by Est1p requires Mg2+.
Figure 4: Mutation in the putative EF hand–like motif affects the G-quadruplex–promoting activity of GSTEst1p.
Figure 5: The loss-of-function mutants of est1 are defective in telomere maintenance.
Figure 6: Extensive telomere elongation is compromised in Est1p mutants that are defective in promoting G-quadruplex formation.
Figure 7: Model of Est1p activating telomere-bound telomerase.

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Acknowledgements

We are grateful to Y. Tsukamoto (Iwate College of Nursing), D. Gottschling (Fred Hutchinson Cancer Research Center), S.-C. Teng (Department of Microbiology, College of Medicine, National Taiwan University), V. Zakian (Department of Molecular Biology, Princeton University) and V. Lundblad (The Salk Institute for Biological Studies) for providing the plasmids and yeast strains. We thank L.-X. Xu for antibody preparations, B.A. Lenzmeier and J. Peng for their critical reading of our manuscript and Y. Hu for creating the image of Figure 7. This work is supported by a Chinese Academy of Sciences–Max Planck Society Professorship and by grants from the Natural Science Foundation of China (NSFC30630018) and the Ministry of Science and Technology (2005CB522402/2007CB914502) to J.-Q.Z.

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M.-L.Z. and X.-J.T. proposed the project, performed most of the experiments, interpreted the data, designed experiments and wrote the manuscript; X.-H.F, B.O.Z., J.W., X.-H.L and Q.-J.L performed some of the experiments; N.S. and J.D. helped with the structure modeling; J.-Q.Z. supervised the project, designed the experiments, interpreted the data and wrote the manuscript.

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Correspondence to Jin-Qiu Zhou.

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Zhang, ML., Tong, XJ., Fu, XH. et al. Yeast telomerase subunit Est1p has guanine quadruplex–promoting activity that is required for telomere elongation. Nat Struct Mol Biol 17, 202–209 (2010). https://doi.org/10.1038/nsmb.1760

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