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TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA

Nature volume 471, pages 532–536 (2011)Cite this article

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Abstract

Maintenance of telomeres requires both DNA replication and telomere ‘capping’ by shelterin. These two processes use two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomeres 1 (POT1). Although RPA and POT1 each have a critical role at telomeres, how they function in concert is not clear. POT1 ablation leads to activation of the ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase at telomeres1,2, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. Unexpectedly, we found that purified POT1 and its functional partner TPP1 are unable to prevent RPA binding to telomeric ssDNA efficiently. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) recapitulates the RPA displacing activity. The RPA displacing activity is inhibited by the telomeric repeat-containing RNA (TERRA) in early S phase, but is then unleashed in late S phase when TERRA levels decline at telomeres3. Interestingly, TERRA also promotes POT1 binding to telomeric ssDNA by removing hnRNPA1, suggesting that the re-accumulation of TERRA after S phase helps to complete the RPA-to-POT1 switch on telomeric ssDNA. Together, our data suggest that hnRNPA1, TERRA and POT1 act in concert to displace RPA from telomeric ssDNA after DNA replication, and promote telomere capping to preserve genomic integrity.

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Figure 1: A novel telomere-specific RPA displacing activity in human cell extracts.
Figure 2: RPA displacement by hnRNPA1.
Figure 3: Regulation of RPA displacement by TERRA.
Figure 4: hnRNPA1 and POT1 suppress the accumulation of RPA at telomeres.

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Acknowledgements

We thank T. de Lange, A. Krainer, B. Chabot and M. Wold for reagents, and members of the Zou laboratory for discussion. L.Z. is an Ellison New Scholar on Aging. R.L.F. is supported by National Institutes of Health (NIH) fellowship 5T32CA009216-28 and American Cancer Society fellowship 0902501. R.C.C. is supported by NIH fellowship F32-GM089150. R.J.O. is supported by the George E. Hewitt Foundation for Medical Research. This work is supported by a Welch Foundation grant (Q-1673) to Z.S., an ACS grant (RSG-08-297) to L.Z., and NIH grants CA133249 to Z.S., CA129037 to S.C., GM06525 and AG025837 to J.K. and GM076388 to L.Z.

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Author notes

  1. Richard C. Centore, Roderick J. O’Sullivan and Rekha Rai: These authors contributed equally to this work.

Authors and Affiliations

  1. Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, 02129, Massachusetts, USA

    Rachel Litman Flynn, Richard C. Centore, Alice Tse & Lee Zou

  2. Department of Pathology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Lee Zou

  3. Molecular and Cellular Laboratory, The Salk Institute for Biological Studies, La Jolla, 92037, California, USA

    Roderick J. O’Sullivan & Jan Karlseder

  4. Department of Laboratory Medicine, Yale University School of Medicine, New Haven, 06510, Connecticut, USA

    Rekha Rai & Sandy Chang

  5. Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, 77030, Texas, USA

    Zhou Songyang

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Contributions

R.L.F. and L.Z. conceived the project. R.L.F., R.C.C., R.J.O., R.R., A.T. and L.Z. performed the experiments. Z.S. contributed the POT1–TPP1 complex purified from insect cells. R.R. and S.C. performed the combined RNA-fluorescence in situ hybridization and immunostaining analysis. R.J.O. and J.K. performed the RPA chromatin immunoprecipitation and northern blot. R.L.F. and L.Z. wrote the paper.

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Correspondence to Lee Zou.

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Flynn, R., Centore, R., O’Sullivan, R. et al. TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA. Nature 471, 532–536 (2011). https://doi.org/10.1038/nature09772

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