Ku is associated with the telomere in mammals.
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Journal: 1999/December - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 10535943
Abstract:
Telomeres are specialized DNA/protein complexes that comprise the ends of eukaryotic chromosomes. The highly expressed Ku heterodimer, composed of 70 and 80 K(d) subunits (Ku70 and Ku80), is the high-affinity DNA binding component of the DNA-dependent protein kinase. Ku is critical for nonhomologous DNA double-stranded break repair and site-specific recombination of V(D)J gene segments. Ku also plays an important role in telomere maintenance in yeast. Herein, we report, using an in vivo crosslinking method, that human and hamster telomeric DNAs specifically coimmunoprecipitate with human Ku80 after crosslinking. Localization of Ku to the telomere does not depend on the DNA-dependent protein kinase catalytic component. These findings suggest a direct link between Ku and the telomere in mammalian cells.
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Proc Natl Acad Sci U S A 96(22): 12454-12458
PMID: 10535943

Ku is associated with the telomere in mammals

Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545; Department of Microbiology and Immunology University of California, San Francisco, CA 94143-0414; and Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
Present address: Life Sciences Division, Lawrence Berkeley National Laboratory, Mail stop 74, 1 Cyclotron Road, Berkeley, CA 94720.
To whom reprint requests should be addressed. E-mail: vog.lbl@nehcjd.
Edited by Joseph G. Gall, Carnegie Institution of Washington, Baltimore, MD, and approved August 31, 1999
Edited by Joseph G. Gall, Carnegie Institution of Washington, Baltimore, MD, and approved August 31, 1999
Received 1999 Jun 14
Copyright © 1999, The National Academy of Sciences

Abstract

Telomeres are specialized DNA/protein complexes that comprise the ends of eukaryotic chromosomes. The highly expressed Ku heterodimer, composed of 70 and 80 Kd subunits (Ku70 and Ku80), is the high-affinity DNA binding component of the DNA-dependent protein kinase. Ku is critical for nonhomologous DNA double-stranded break repair and site-specific recombination of V(D)J gene segments. Ku also plays an important role in telomere maintenance in yeast. Herein, we report, using an in vivo crosslinking method, that human and hamster telomeric DNAs specifically coimmunoprecipitate with human Ku80 after crosslinking. Localization of Ku to the telomere does not depend on the DNA-dependent protein kinase catalytic component. These findings suggest a direct link between Ku and the telomere in mammalian cells.

Abstract

Vertebrate telomeric DNA is composed of (T2AG3) tandem repeats, with the number of repeats varying between different species (1, 2). The GT-rich strand of telomeric DNA is synthesized by telomerase, a specialized ribonucleoprotein reverse transcriptase. A minimal telomeric DNA length and, in some situations, an active telomerase are required for chromosome stability and cellular viability (3, 4), with failure to maintain telomere length or function leading to a form of replicative senescence in Tetrahymena, yeasts, and mammalian cells (5–7). Activation of telomerase is characteristic of most established mammalian cell lines and tumors (8). Correspondingly, experimental activation of telomerase also allows certain virus-transformed human cell lines to bypass authentic cellular replicative senescence and crises and continue proliferation (4, 6, 9).

Several lines of evidence have recently converged supporting the conclusion that the Ku heterodimer is critical for telomere maintenance in yeast. Saccharomyces cerevisiae null for Ku70 or Ku80 have defects in telomere silencing, abnormally short telomeres, and a senescence-like phenotype at 37°C (10–13). Strikingly, despite the relatively low sequence conservation between yeast and human Ku subunits at the amino acid level, the exogenous expression of human Ku subunits rescues Ku null yeast from cell death at 37°C (14). Direct evidence for the physical localization of the Ku heterodimer to yeast telomeres came from an in vivo crosslinking experiment linking Ku to telomeric DNA (15). Recently, Martin et al. (16) demonstrated in yeast that Ku80 colocalizes with the telomere binding protein Rap1 at telomeric foci. These types of experiments have not been possible to perform in mammalian cells because of the large abundance and uniform distribution of Ku throughout the mammalian nucleus.

However, nothing has been reported concerning the Ku heterodimer’s role at the mammalian telomere. Using an in vivo crosslinking method, we show that Ku is localized to mammalian telomeric repeats. We also determined that the DNA-dependent protein kinase catalytic component (DNA-PKcs) is not required in vivo for the association of Ku with telomeric repeats.

Acknowledgments

We thank Joan Tuner for providing MO59J and MO59K cells lines; Ning-Hsin Yeh for generously supplying anti-Ku80 hybridoma; and Sandeep Burma, Judith Campisi, and Priscilla Cooper for critical reading of the manuscript and for helpful suggestions. This work was supported by the U.S. Department of Energy and by National Institutes of Health Grants CA50519 (to D.J.C.) and GM26259 (to EHB).

Acknowledgments

Abbreviations

DNA-PKcsDNA-dependent protein kinase catalytic component
HSFhuman skin fibroblast
CHOChinese hamster ovary
Abbreviations

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

Footnotes

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