In addition to the classical DNA-PK-dependent NHEJ (C-NHEJ) repair of DSBs, numerous recent results have brought evidence for the existence of alternative end-joining mechanisms or Back-up NHEJ (B-NHEJ) which may be particularly relevant to genomic instability associated with cancer. For example, they appear highly mutagenic through frequent non-reciprocal translocations leading to a high level of lymphomagenesis and other cancers in C-NHEJ deficient animal models. In addition, reciprocal chromosome translocations like the ones at the origin of leukemia are mediated by a rejoining pathway which shows signatures of an error-prone alternative NHEJ pathway. Thus, deciphering the components and mechanisms of these pathways is a key step towards understanding tumorigenesis.
B-NHEJ is mostly repressed by Ku under normal condition. Several protein candidates have been proposed to participate in these pathways. In addition, biochemical assays with cell extracts in our and other groups have shown that Ku compete with PARP1 for DNA end-binding, that PARP1 can perform a synapsis activity, that PARP1 activity is required for a subsequent XRCC1/ligase III joining step and that PNK can assist the reaction. However, more efforts are needed to identify all the actors of these pathways.
Our aim is to characterize biochemically the proteins involved in alternative routes for end-joining of DNA DSBs in human cells. We use biochemical approaches in cellulo and in vitro based on minimizing the inhibitory effect of Ku.