Single molecule super-resolution mapping of the spatiotemporal organization of DNA double strand break repair

Whelan DR1,2, Lee WTC2, Yin Y2, Fenyo D2 and Rothenberg E2

  1. La Trobe Institute for Molecule Science, La Trobe University, Edwards Road, Flora Hill, Victoria, Australia, 3552.
  2. Department of Biochemistry and Molecular Pharmacology, Langone Medical Center, New York University, 550 First Ave, New York, NY, USA, 10016.

DNA damage response (DDR) pathways are involved in both the cause and potential treatment of various cancers, auto-immune, and neurodegenerative diseases. Our current understanding of DDR has been elucidated over the course of several decades by combining biochemical and biophysical techniques, however, imaging of damage and repair in vivo has remained challenging. This has predominantly been because of the dense and varied nature of the nuclear environment, and the diffraction limit of light. Here, we have successfully used single molecule localisation super resolution (SR) imaging to circumvent this limit and capture spatially and temporally resolved snapshots of double strand break (DSB) repair in cells. Moreover, we specifically generated individual single-ended DSBs similar to those endogenously created by collapsing replication forks. The resulting repair foci could be visualized in multicolor SR by labelling nascent DNA via modified base incorporation and click chemistry, DSBs via the TUNEL assay or direct ligation, single stranded DNA via BrdU incorporation, and proteins via immunolabelling. The enhanced spatial and temporal resolutions and the singular nature of the DSBs themselves revealed several exciting and novel insights including the dynamic interactions of proteins such as Ku, MRE11 and RAD51 at the DSB, the redundant role of RAD52 in repair, and a critical in vivo BRCA2 dependence on BRCA1. I will present these findings within the context of their importance to the genomic integrity research community, as well as the broader novelty and applicability of the SR assays we have developed.