Single-molecule studies reveal mechanisms of human DNA double-strand break repair

DNA damage is ubiquitous to all organisms and very complex pathways have evolved to recognize and repair these lesions. The most deleterious DNA damages are double-strand breaks (DSBs), and a single unrepaired DSB can lead to cell death. In human cells, there exist two canonical pathways of DSB repair: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Two protein complexes that rapidly bind DNA ends coordinate these separate pathways: the Ku70-Ku80 heterodimer (Ku) and the Mre11-Rad50-Nbs1 complex (MRN), respectively. Ku encircles the DNA ends and recruits other factors, such the kinase DNA-PKcs, to bluntly ligate the ends back together. In contrast, MRN along with the long-range nuclease Exo1 and helicase BLM digests the DNA to create long 3’ single-stranded DNA overhangs, which are rapidly bound by the single-stranded DNA binding protein RPA. Next, Rad51 replaces RPA and facilitates strand exchange into a homologous chromosome to resynthesize the missing information in a largely error-free way. Despite the importance of DSB repair, many of the underlying mechanisms by which these molecular machines dynamically assemble and carry out the repair process have remained unknown. Here, I use a combination of ensemble biochemical assays as well as high-throughput single-molecule microscopy to visualize the repair process. I have observed two main steps of the repair process: initiation of HR by MRN and long-range resection by Exo1. I have found that MRN locates DSBs by a sliding mechanism that allows it to load on Ku-blocked ends. Then, once it reaches the end, MRN removes DNA-PK and recruits Exo1 and BLM in order to promote long-range digestion of the DNA. Finally, the Exo1/BLM resectosome is attenuated by phosphorylation of RPA. Overall, I have characterized the initiation and regulation of DSBR. This will lead to a new understanding of the ways in which these deleterious lesions are repaired and will contribute to understanding cancer as well as techniques for genetic manipulation.