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Ku stimulation of DNA ligase IV-dependent ligation requires inward movement along the DNA molecule
journal contribution
posted on 2023-06-08, 07:44 authored by Boris Kysela, Aidan DohertyAidan Doherty, Miroslav Chovanec, Thomas StiffThomas Stiff, Simon M Ameer-Beg, Borivoj Vojnovic, Pierre-Marie Girard, Penny JeggoThe 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.
History
Publication status
- Published
Journal
Journal of Biological ChemistryISSN
0021-9258Publisher
American Society for Biochemistry and Molecular BiologyExternal DOI
Issue
25Volume
278Page range
22466-22474Pages
9.0Department affiliated with
- Sussex Centre for Genome Damage Stability Publications
Full text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2012-02-06Usage metrics
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