22nd Annual NASA Space Radiation Investigators' Workshop (2011)
7037.pdf
Tankyrase 1-Dependent pADPr Modification of DNA-PKcs is Essential
for Holoenzyme Formation and Function
R. C. Dregalla, D. G. Maranon, H. L. Liber and S. M. Bailey
Department of Environmental & Radiological Health Sciences
Colorado State University, Fort Collins, CO 80523-1618
Intrigued by the dynamics of the seemingly contradictory yet integrated cellular responses to the
requisites of preserving telomere integrity while also efficiently repairing damaged DNA, we investigated
roles of the telomere associated poly(adenosine diphosphate [ADP]‐ribose) polymerase (PARP) tankyrase
1 in both telomere function and the DNA damage response following exposure to ionizing radiation [1].
Tankyrase 1 siRNA knockdown in human cells significantly elevated recombination specifically within
telomeres, hotspots for sister chromatid exchange (T‐SCE), a phenotype that quantitative modeling
suggests has the potential of accelerating cellular senescence [2, 3]. We have also observed high T‐SCE
rates in cells with deficiencies in WRN and BLM, the genes defective in Werner’s and Bloom’s
syndromes, supporting a connection to premature aging [4].
Much more unexpected was the observation that depletion of tankyrase 1 resulted in concomitant and
rapid reduction of the nonhomologous end‐joining (NHEJ) protein DNA dependent protein kinase
catalytic subunit (DNA‐PKcs), while Ku86 and ATM protein levels remained unchanged; DNA‐PKcs
mRNA levels were also unaffected. We demonstrated that depletion of tankyrase 1 resulted in
proteasome‐mediated DNA‐PKcs degradation, thereby explaining the associated defective damage
response observed; i.e., increased sensitivity to ionizing radiation‐induced cell killing, mutagenesis,
chromosome aberration and telomere fusion, regardless of radiation quality. These studies were
consistent with identification of DNA-PKcs amongst poly(ADP-ribose) protein complexes [5], and
provided the first evidence for regulation of DNA-PKcs by tankyrase 1 PARP activity [1, 6]. However,
any potential role of PARsylated DNA-PKcs in processes such as NHEJ remained to be elucidated. Here,
we investigated the role of PARsylated DNA-PKcs in NHEJ-mediated double-strand break (DSB) repair
in human cells. We found that tankyrase 1 PARP catalytic activity is required for recruitment to and
function of DNA-PKcs at ionizing radiation-induced DSB sites, and so provide the first insights into the
relevance of regulation of DNA-PKcs protein stability by pADPr modification. Our results demonstrate
that inhibition of telomere-associated tankyrase 1 catalytic activity compromises DSB-repair kinetics as a
consequence of abrogated DNA-PK holoenzyme assembly, and so provide new understanding into basic
mechanisms of DNA repair following radiation injury. We propose a model in which the stepwise
pADPr modification of DNA-PK subunits provides plausible explanation for the ‘DNA-dependent’ nature
of the functional holoenzyme.
The authors gratefully acknowledge support for this research from NASA (NNJ04HD83G and
NNX08AB65G).
References
1.
Dregalla, R.C., et al., Regulatory roles of tankyrase 1 at telomeres and in DNA repair:
suppression of T-SCE and stabilization of DNA-PKcs. Aging, 2010. 2(10): p. 691-708.
2.
Blagoev, K.B., E.H. Goodwin, and S.M. Bailey, Telomere sister chromatid exchange and the
process of aging. Aging, 2010. 2(10): p. 727-30.
3.
Blagoev, K.B. and E.H. Goodwin, Telomere exchange and asymmetric segregation of
chromosomes can account for the unlimited proliferative potential of ALT cell populations. DNA
Repair (Amst), 2008. 7(2): p. 199-204.
4.
Hagelstrom, R.T., et al., Hyper telomere recombination accelerates replicative senescence and
may promote premature aging. Proc Natl Acad Sci U S A, 2010. 107(36): p. 15768-73.
5.
Gagne, J.P., et al., Proteome-wide identification of poly(ADP-ribose) binding proteins and
poly(ADP-ribose)-associated protein complexes. Nucleic Acids Res, 2008. 36(22): p. 6959-76.
6.
Chang, S., The telomere protein tankyrase 1 regulates DNA damage responses at telomeres.
Aging, 2010. 2(10): p. 639-42.