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Human telomeric protein TRF2 associates with genomic double-strand breaks as an early response to DNA damage

Nature Genetics volume 37, pages 193–197 (2005)Cite this article

Abstract

DNA damage surveillance networks in human cells can activate DNA repair, cell cycle checkpoints and apoptosis in response to fewer than four double-strand breaks (DSBs) per genome1,2. These same networks tolerate telomeres, in part because the protein TRF2 prevents recognition of telomeric ends as DSBs3 by facilitating their organization into T loops4,5,6. We now show that TRF2 associates with photo-induced DSBs in nontelomeric DNA in human fibroblasts within 2 s of irradiation. Unlike γH2AX, a common marker for DSB damage, TRF2 forms transient foci that colocalize closely with DSBs. The TRF2 DSB response requires the TRF2 basic domain but not its Myb domain and occurs in the absence of functional ATM and DNA-PK protein kinases, MRE11/Rad50/NBS1 complex and Ku70, WRN and BLM repair proteins. Furthermore, overexpression of TRF2 inhibits DSB-induced phosphorylation of ATM signaling targets. Our results implicate TRF2 in an initial stage of DSB recognition and processing that occurs before association of ATM with DSBs and activation of the ATM-dependent DSB response network.

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Figure 1: TRF2 associates with photoinduced γH2AX foci in SV40-transformed human fibroblasts (GM00639).
Figure 2: Colocalization of TRF2 and ATM foci with photoinduced DSBs ∼10 min after laser irradiation.
Figure 3: Live-cell imaging of fluorescently tagged TRF2 proteins after photoinduction of DSBs in GM00639 fibroblasts shows differences in recruitment of YFP-TRF2, GFP-TRF2-ΔM and GFP-TRF2-ΔB to regions of photoinduced DSBs in individual nuclei.
Figure 4: The kinetics of TRF2 association with photoinduced DSBs in GM00639 fibroblasts.
Figure 5: Association of TRF2 with photoinduced DSBs in mutant fibroblasts.
Figure 6: Overexpression of TRF2 in primary human fibroblasts attenuates the ATM-dependent cellular response to DSBs.

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Acknowledgements

We thank D. Chen for Ku70-deficient G22p1−/− fibroblasts and T. de Lange for the AdTRF2 vector and for advice. This work was supported by a Canadian Institutes for Health Research/Fanconi Anaemia Canada Fellowship Award (to P.S.B.) and an operating grant from the National Cancer Institute of Canada (to M.S.M.). D.J.S. is supported by Ontario Graduate and Hospital for Sick Children Research Institute Restracomp Scholarships.

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Author notes

  1. Paul S Bradshaw and Dimitrios J Stavropoulos: These authors contributed equally to this work.

Authors and Affiliations

  1. Program in Genetics and Genomic Biology, Research Institute, The Hospital for Sick Children, Toronto, M5G 1X8, Ontario, Canada

    Paul S Bradshaw, Dimitrios J Stavropoulos & M Stephen Meyn

  2. Department of Molecular and Medical Genetics, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    Paul S Bradshaw, Dimitrios J Stavropoulos & M Stephen Meyn

  3. Department of Paediatrics, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    M Stephen Meyn

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  1. Paul S Bradshaw
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  2. Dimitrios J Stavropoulos
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  3. M Stephen Meyn
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Correspondence to M Stephen Meyn.

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Supplementary information

Supplementary Fig. 1

TRF2 associates with tracks of photo-induced DSBs in primary human fibroblasts (GM45043), as detected by indirect immunofluorescence analysis of cells fixed ∼10 minutes post-irradiation. (PDF 43 kb)

Supplementary Fig. 2

GFP-TRF2-ΔM localizes to telomeres in unirradiated human fibroblasts. (PDF 185 kb)

Supplementary Fig. 3

Telomeric repeat DNA does not accumulate in chromatin domains containing photo-induced DSBs in irradiated human fibroblasts. (PDF 297 kb)

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Bradshaw, P., Stavropoulos, D. & Meyn, M. Human telomeric protein TRF2 associates with genomic double-strand breaks as an early response to DNA damage. Nat Genet 37, 193–197 (2005). https://doi.org/10.1038/ng1506

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