title: Conformational transitions of proteins engaged in DNA double-strand break repair, analysed by tryptophan fluorescence emission and FRET.
abstract: 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.
authors.affiliation: National Center of Radiobiology and Radiation Protection, Georgi Sofiyski 3, 1606 Sofia, Bulgaria.
authors.forename: Katia
authors.initials: K
authors.lastname: Stankova
authors.forename: Katia
authors.initials: K
authors.lastname: Ivanova
authors.forename: Emil
authors.initials: E
authors.lastname: Mladenov
authors.forename: Bustanur
authors.initials: B
authors.lastname: Rosidi
authors.forename: Aparna
authors.initials: A
authors.lastname: Sharma
authors.forename: Rayna
authors.initials: R
authors.lastname: Boteva
authors.forename: George
authors.initials: G
authors.lastname: Iliakis
mesh_terms: D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D011506:Proteins; D014364:Tryptophan