Ku stimulation of DNA ligase IV-dependent ligation requires inward movement along the DNA molecule

Kysela, Boris, Doherty, Aidan J, Chovanec, Miroslav, Stiff, Thomas, Ameer-Beg, Simon M, Vojnovic, Borivoj, Girard, Pierre-Marie and Jeggo, Penny A (2003) Ku stimulation of DNA ligase IV-dependent ligation requires inward movement along the DNA molecule. Journal of Biological Chemistry, 278 (25). pp. 22466-22474. ISSN 0021-9258

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The DNA ligase IXRCC4 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.

Item Type: Article
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Depositing User: Aidan Doherty
Date Deposited: 06 Feb 2012 20:46
Last Modified: 19 Mar 2013 14:03
URI: http://sro.sussex.ac.uk/id/eprint/28010
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