RubicKu

The Ku70-Ku80 heterodimer (KU) is a core factor of the NHEJ (Non-homologous end joining) repair pathway. It recognizes DNA ends formed upon double-strand break (DSBs), contributes to tether proximal DNA ends, recruits nucleases and DNA polymerases for ends processing when needed and positions the ligase for final repair. KU has emerged as a central factor for several additional genome stability pathways including replication fork processing and telomeres maintenance. During the last years, the 3 partners of the RubicKU consortium initiated collaborations to understand the molecular basis of the multifaceted roles of KU using two complementary model organisms, the fission yeast S. pombe and the budding yeast S. cerevisae. P1 and P2 thus showed that KU binds DNA:RNA hybrid at stalled replication fork and fine tune fork restart. P1 and P3 characterized the protective roles of KU at telomeres ends. In this project, we will combine a large set of complementary state-of-the-art structural, biochemical and genetic approaches to disentangle the molecular determinant allowing to specific KU functions in distinct genome stability pathways. First, we will characterize at the atomic level the interactions of KU with DNA, RNA and DNA:RNA substrates and characterize KU mutants selectively impaired for one of these substrates binding (WP1). Mutants will be identified through structural analysis and genetic screens. We will reconstitute and structurally characterize yeast NHEJ complexes and assemblies and compare with human assemblies (recently determined by P1) (WP2). We will disentangle the molecular determinants of KU functions at replication forks and show how its binding to RNA:DNA hybrids regulate nascent strand degradation and fine-tune fork stability (WP3). We will characterize the unexplored interaction of KU with the constitutive DNA:RNA hybrids generated by fill-in synthesis at telomere ends during the last steps of telomere replication (WP4).