Characterization of the macromolecular interactions of the key NHEJ components

Costantini, Silvia Elvira (2007). Characterization of the macromolecular interactions of the key NHEJ components. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000fb0f

Abstract

Non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double strand breaks in mammalian cells. Six ‘core’ NHEJ components have been identified to date: Ku70, Ku80, the DNA dependent protein kinase catalytic subunit DNA-PKcs, DNA ligase IV, XRCC4, and XLF-Cemunnos. In this thesis, the DNA binding properties of the Ku70/80 heterodimer are characterised by means of fluorescence anisotropy and electrophoretic mobility shift assays. Studies with duplexes holding 1, 2 or 3 heterodimers allowed to develop a binding model that can be applied to analyse the interactions of Ku with duplexes of any length. In addition, salt dependent studies indicate that electrostatic interactions play a major role in the binding of Ku to nucleic acids. The interaction of the Ku heterodimer with the DNA ligase IV/XRCC4 (LX) complex and the role of DNA-PKcs in regulating the NHEJ complex
assembly are investigated. Protein-protein interaction experiments show that Ku interacts with DNA ligase IV via its tandem BRCT domain and this interaction is enhanced by the presence of XRCC4 and DNA. In particular, mutagenesis studies indicate that residues 643-748 encompassing the first BRCT domain of DNA ligase IV are necessary for binding. Moreover, Ku needs to be in its heterodimeric form to bind LX and the C-terminal of Ku80 is dispensable for this interaction. The presence of DNA-PKcs favours the interaction between Ku and LX, although its kinase activity induces the disassembly of the complex. Finally, the interaction of Ku with other factors potentially involved in NHEJ such as the replication protein A, PARP-1, and the WRN helicase is investigated. Collectively, these findings provide novel information on the macromolecular interactions that regulate the NHEJ pathway.

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