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J. Biol. Chem., Vol. 278, Issue 25, 22466-22474, June 20, 2003

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Ku Stimulation of DNA Ligase IV-dependent Ligation Requires Inward Movement along the DNA Molecule^*

Boris Kysela  , Aidan J. Doherty ¶ ||, Miroslav Chovanec  **, Thomas Stiff , Simon M. Ameer-Beg , Borivoj Vojnovic , Pierre-Marie Girard  and Penny A. Jeggo  

From the Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, the ^¶Cambridge Institute for Medical Research and Department of Haematology, University of Cambridge, Hill Rd., Cambridge CB2 2XY, and the Gray Cancer Institute, P. O. Box 100, Mount Vernon Hospital, Northwood, Middlesex, HA6 2JR, United Kingdom

The DNA ligase IV·XRCC4 complex (LX) functions in DNA non-homologous-end joining, the main pathway for double-strand break repair in mammalian cells. We show that, in contrast to ligation by T4 ligase, the efficiency of LX ligation of double-stranded (ds) ends is critically dependent upon the length of the DNA substrate. The effect is specific for ds ligation, and LX/DNA binding is not influenced by the substrate length. Ku stimulates LX ligation at concentrations resulting in 1–2 Ku molecules bound per substrate, whereas multiply Ku-bound DNA molecules inhibit ds ligation. The combined footprint of DNA with Ku and LX bound is the sum of each individual footprint suggesting that the two complexes are located in tandem at the DNA end. Inhibition of Ku translocation by the presence of cis-platinum adducts on the DNA substrate severely inhibits ligation by LX. Fluorescence resonance energy transfer analysis using fluorophore-labeled Ku and DNA molecules showed that, as expected, Ku makes close contact with the DNA end and that addition of LX can disrupt this close contact. Finally, we show that recruitment of LX by Ku is impaired in an adenylation-defective mutant providing further evidence that LX interacts directly with the DNA end, possibly via the 5'-phosphate as shown for prokaryotic ligases. Taken together, our results suggest that, when LX binds to a Ku-bound DNA molecule, it causes inward translocation of Ku and that freedom to move inward on the DNA is essential to Ku stimulation of LX activity.

Received for publication, March 31, 2003

^* This work was supported in part by grants from the Medical Research Council, the Human Frontiers Science Program, the Leukemia Research Fund, the Primary Immunodeficiency Association, and the European Union Grant Figh CT 1999 (to the P. A. J. laboratory); by grants from Cancer Research UK, the Association for International Cancer Research, the Biotechnology and Biological Sciences and Research Council, and the Royal Society (to the A. J. D. laboratory); and by Cancer Research UK under Program Grant C133/A1812 and the Royal Society Paul Instrument Fund (to the B. V. laboratory). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| A Royal Society University Research Fellow.

^** Recipient of a Royal Society/NATO Postdoctoral Fellowship grant. On leave from the Laboratory of Molecular Genetics, Cancer Research Institute, Vlarska 7, 833 91 Bratislava 37, Slovak Republic.

To whom correspondence may be addressed. Tel.: 44-12-73-678-482; Fax: 44-12-73-678-121; E-mail: p.a.jeggo{at}sussex.ac.uk (P. A. J.) or b.kysela{at}sussex.ac.uk (B. K.).

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