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Distinct faces of the Ku heterodimer mediate DNA repair and telomeric functions

Nature Structural & Molecular Biology volume 14, pages 301–307 (2007)Cite this article

Abstract

The Ku heterodimer, comprised of Ku70 and Ku80 subunits, is a conserved complex involved in nonhomologous end-joining (NHEJ). However, it also functions in maintenance of telomeres, chromosome termini normally resistant to end-joining events. To elucidate the spatial organization of these functions, we rationally guided Ku mutagenesis in yeast with real-valued evolutionary trace (rvET). This revealed two ancestrally related α-helices: one on the Ku70 surface that is required in yeast for NHEJ, and a second on the Ku80 surface that is required in yeast for telomeric heterochromatin formation. When bound to a DNA end, the surface containing the NHEJ-specific Ku70 helix is oriented toward the DNA terminus, whereas the surface containing the telomeric function–specific Ku80 helix faces inward, toward telomeric chromatin, when bound to a telomere. We propose a 'two-face' model for Ku and that divergent evolution of these faces allowed Ku's dual role in NHEJ and telomere maintenance.

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Figure 1: The Yku80 vWA evolutionary trace cluster is required for Ku's telomeric functions.
Figure 2: Yku80 surface α-helix 5 is essential for telomeric silencing.
Figure 3: Yeast Ku80 α5 mutations impair Sir4-Yku80 interaction, as measured by the activity of a β-galactosidase reporter (see Methods).
Figure 4: Yku70 surface α-helix 5 is essential for NHEJ.
Figure 5: Two-face model of Ku's functional organization at DSBs compared with telomeres.

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Acknowledgements

We thank J. Huang and D. Wuttke for critical reading of the manuscript, S. Almaguer, S. Haricharan and C. Williams for technical assistance and H. Feldmann (Universität Munchen), D. Gottschling (Fred Hutchinson Cancer Research Center), J. Haber (Brandeis University) and V. Lundblad (Salk Institute for Biological Studies) for strains and plasmids. This work was supported by US National Institutes of Health grant RO1-GM066099, US National Science Foundation grant DBI0547695 and the March of Dimes grant FY06-371 (O.L.), and by a gift from the Cyvia and Melvyn Wolff Family Foundation (A.A.B.). rvET analysis is available publicly through the Lichtarge Computational Biology Lab server (http://mammoth.bcm.tmc.edu/).

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Authors and Affiliations

  1. Department of Pediatrics, Hematology/Oncology Section, Baylor College of Medicine, One Baylor Plaza, BCM225, Houston, 77030-3411, Texas, USA

    Albert Ribes-Zamora & Alison A Bertuch

  2. Program in Cell & Molecular Biology, Baylor College of Medicine, One Baylor Plaza, BCM225, Houston, 77030-3411, Texas, USA

    Albert Ribes-Zamora

  3. Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, BCM225, Houston, 77030-3411, Texas, USA

    Ivana Mihalek & Olivier Lichtarge

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Contributions

A.R.-Z. was the first to propose using evolutionary trace to study the Ku heterodimer. He performed all of the experiments except for the telomeric single-stranded overhang analysis, and, together with A.A.B., conceived the experimental design and interpreted the data. I.M. performed the rvET analysis and contributed to data analysis. O.L. oversaw the rvET analysis and contributed to data analysis. A.A.B. performed the telomeric single-stranded overhang analysis and oversaw the entire project. All authors contributed to the preparation of the final manuscript.

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Correspondence to Alison A Bertuch.

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Supplementary information

Supplementary Fig. 1

Yku80 mutants with global telomere defects. (PDF 2695 kb)

Supplementary Fig. 2

G-tail analysis of yku80 α5 mutant strains. (PDF 927 kb)

Supplementary Table 1

Summary of Ku80 vWA-like cluster mutagenesis. (PDF 91 kb)

Supplementary Table 2

Summary of Ku70 vWA-like cluster mutagenesis. (PDF 68 kb)

Supplementary Table 3

Yeast strains. (PDF 95 kb)

Supplementary Table 4

Plasmids. (PDF 74 kb)

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Ribes-Zamora, A., Mihalek, I., Lichtarge, O. et al. Distinct faces of the Ku heterodimer mediate DNA repair and telomeric functions. Nat Struct Mol Biol 14, 301–307 (2007). https://doi.org/10.1038/nsmb1214

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