Achieving Selectivity in Space and Time with DNA Double-Strand-Break Response and Repair: Molecular Stages and Scaffolds Come with Strings Attached (360G-Wellcome-200814_Z_16_Z)

Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) are the two major pathways of DNA double-strand break (DSB) repair in human cells. The aim of my research is to understand how NHEJ repairs DSBs directly without a DNA template but with maximal selectivity. Biochemical, structural and functional studies of individual components and complexes involved in NHEJ, carried out in my lab and elsewhere, suggest that several mechanisms operate throughout synapsis, end processing and ligation to maintain correct co- localisation of components over time. These are: (i) a stage provided by Ku- heterodimer interacting with DSBs supporting DNA-PKcs, APLF, BRCA1, PAXX amongst others; (ii) a second stage, DNA-PKcs, which links the kinase with DNA, Ku, PARP1, BRCA1 and Artemis; (iii) a temporary scaffold, which facilitates repair operations, constructed from XRCC4-XLF filaments, assembling to bridge Ku bound at DSB ends. Lig IV bound to XRCC4 C-termini likely terminates the scaffold, bringing LigIV close to DNA broken ends; (iv) a string provided by Artemis C-terminal-extension, which is intrinsically disordered, but includes short inear "epitopes" that recognise DNA-PKcs, DNA LigIV and PTIP, and keeps components closeby. My specific objectives are to study how these different but complementary ways provide colocalisation and efficient DSB repair.