Mechanisms of Non-Homologous DNA End Joining:Aspects of In Vitro Assays

Abstract

Double-strand breaks (DSB) in genomic DNA are a major threat to cell survival and chromosome integrity. In vertebrate cells, non-homologous DNA end joining (NHEJ) is the major pathway of DSB repair. Genetic studies in yeast, human and rodent cell lines displaying increased IR sensitivity and defects in DSB repair have provided insight in the genes involved in NHEJ. These genetic data have been confirmed and complemented by in vitro assays which have played a significant role in the elucidation and the dissection of the basic mechanisms underlying NHEJ. In vitro assays utilize model DNA substrates that carry defined DSB and thus provide information on the efficiency and fidelity of NHEJ in different cell systems. In contrast to investigations in living cells, in vitro assays facilitate the investigation of the functions of single proteins in the repair process itself so that their impact on the rejoining of different DNA end structures can be studied directly without interference by other cellular processes such as cell cycle and replication. In this chapter, we summarize the basic features of in vitro assays and give an overview over the different available cell-free systems which have facilitated the detailed analysis of NHEJ mechanisms in different vertebrate cells.