Studying DNA Double-Strand Break Repair: An Ever-Growing Toolbox

To ward off against the catastrophic consequences of persistent DNA double-strand breaks (DSBs), eukaryotic cells have developed a set of complex signaling networks that detect these DNA lesions, orchestrate cell cycle checkpoints and ultimately lead to their repair. Collectively, these signaling networks comprise the DNA damage response (DDR).

Our capacity to create DSBs in a programed manner and in such a way that is compatible with a set of diverse methodologies to investigate the events that follow DNA damage, has led to our current deep understanding of the DDR. The induction of DSBs at random locations using different sources of radiation or genotoxic compounds, provides the easiest approach to analyze the recruitment kinetics of proteins to sites of DNA damage and is a powerful strategy to temporally resolve the sequence of DNA repair events. The development of methods to induce annotated DNA breaks at transgenic loci inserted in the genome, or at endogenous loci (restriction enzymes, CRISPR/Cas9) allowed the analysis of the DDR at molecular resolution and were instrumental in disclosing functional links between the DDR and processes such as transcriptional, chromatin dynamics, and DNA replication. Yet all the tools described here display significant drawbacks. For instance, nucleases-induced DSBs undergo consecutive cycles of repair/cleavage until these have been mutated, calling for caution when investigating DNA repair using these tools. A major challenge is now to refine these DSB-inducible systems and the subsequent methodologies to analyze repair in order to overcome these limitations.