We analysed protein–DNA and protein–protein interactions relevant to the repair of DNA DSBs (double-strand breaks) by NHEJ (non-homologous end-joining). Conformational transitions in mammalian DNA ligases III (LigIII) and IV (LigIV), as well as in PARP-1 [poly(ADP-ribose) polymerase-1], were analysed upon binding to double-stranded DNA by changes in tryptophan emission and FRET (Förster resonance energy transfer) from tryptophan to DNA-conjugated Alexa Fluor® 532. For LigIII, two non-equivalent high- and low-affinity DNA-binding sites are detected interacting sequentially with DNA. PARP-1 displays a single high-affinity DNA-binding site and can displace bound DNA fragments from the low-affinity site of LigIII, consistent with its mediator role in LigIII–DNA interactions. For the LX [LigIV–XRCC4 (X-ray cross-complementation group 4)] complex, a single DNA-binding site is detected. Binding of Ku to DNA was accompanied by conformational changes in the protein and intermolecular FRET from dansyl chromophores of the labelled Ku to the Alexa Fluor® chromophores of Alexa Fluor® 532-conjugated DNA. The average distance of 5.7 nm calculated from FRET data is consistent with a location of Ku at the very end of the DNA molecule. Binding of LX to Ku–DNA complexes is associated with conformational changes in Ku, translocating the protein further towards the DNA ends. The protein–protein and protein–DNA interactions detected and analysed generate a framework for the characterization of molecular interactions fundamental to the function of NHEJ pathways in higher eukaryotes.
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Research Article|
April 16 2012
Conformational transitions of proteins engaged in DNA double-strand break repair, analysed by tryptophan fluorescence emission and FRET
Katia Stankova;
Katia Stankova
*National Center of Radiobiology and Radiation Protection, Georgi Sofiyski 3, 1606 Sofia, Bulgaria
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Katia Ivanova;
Katia Ivanova
*National Center of Radiobiology and Radiation Protection, Georgi Sofiyski 3, 1606 Sofia, Bulgaria
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Emil Mladenov;
Emil Mladenov
†University of Duisburg-Essen, Medical School, Institute of Medical Radiation Biology, Hufelandstrasse 55, 45122 Essen, Germany
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Bustanur Rosidi;
Bustanur Rosidi
1
†University of Duisburg-Essen, Medical School, Institute of Medical Radiation Biology, Hufelandstrasse 55, 45122 Essen, Germany
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Aparna Sharma;
Aparna Sharma
†University of Duisburg-Essen, Medical School, Institute of Medical Radiation Biology, Hufelandstrasse 55, 45122 Essen, Germany
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Rayna Boteva;
Rayna Boteva
2
*National Center of Radiobiology and Radiation Protection, Georgi Sofiyski 3, 1606 Sofia, Bulgaria
2Correspondence may be addressed to either of these authors (email r.boteva@ncrrp.org or Georg.Iliakis@uk-essen.de).
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George Iliakis
George Iliakis
2
†University of Duisburg-Essen, Medical School, Institute of Medical Radiation Biology, Hufelandstrasse 55, 45122 Essen, Germany
2Correspondence may be addressed to either of these authors (email r.boteva@ncrrp.org or Georg.Iliakis@uk-essen.de).
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Publisher: Portland Press Ltd
Received:
December 13 2011
Revision Received:
February 14 2012
Accepted:
February 17 2012
Accepted Manuscript online:
February 17 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 443 (3): 701–709.
Article history
Received:
December 13 2011
Revision Received:
February 14 2012
Accepted:
February 17 2012
Accepted Manuscript online:
February 17 2012
Citation
Katia Stankova, Katia Ivanova, Emil Mladenov, Bustanur Rosidi, Aparna Sharma, Rayna Boteva, George Iliakis; Conformational transitions of proteins engaged in DNA double-strand break repair, analysed by tryptophan fluorescence emission and FRET. Biochem J 1 May 2012; 443 (3): 701–709. doi: https://doi.org/10.1042/BJ20112151
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