Poster Presentation 43rd Lorne Genome Conference 2022

Mapping DNA repair complexes at stalled and broken replication forks using super resolution microscopy (#275)

Donna R Whelan 1 , Toby DM Bell 2 , Eli Rothenberg 3
  1. La Trobe University, Flora Hill, VIC, Australia
  2. Monash University, Clayton, Victoria, Australia
  3. School of Medicine, New York University, New York, USA

DNA damage response (DDR) pathways are involved in both the cause and 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. We have successfully developed dSTORM super resolution (SR) imaging assays that circumvent this limit and capture spatially and temporally resolved snapshots of individual replication forks and associated stress and damage in cells. These replication and repair foci can be visualized in multicolor SR by labelling nascent DNA by modified base incorporation and click chemistry, DSBs by the TUNEL assay or direct ligation, single stranded DNA by BrdU incorporation, and proteins by immunolabelling [1]. The enhanced spatial and temporal resolutions and the singular nature of the forks and DSBs has uncovered several novel insights including the dynamic interactions of proteins such as Ku, MRE11 and RAD51 at the DSB [2], the redundant role of RAD52 in repair, and a critical in vivo BRCA2 dependence on BRCA1 [3]. We have also extended these assays to investigate the roles of long non-coding RNA and several novel proteins in DSB repair. 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.

  1. 1. Whelan, D.R., et al., Spatiotemporal dynamics of homologous recombination repair at single collapsed replication forks. Nature Communications, 2018. 9(1): 3882.
  2. 2. Whelan, D.R. and E. Rothenberg, Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination. PNAS, 2021. 118(11): e2021963118.
  3. 3. Whelan, D.R., et al., Super-resolution visualization of distinct stalled and broken replication fork structures. Plos Genetics, 2020 16(12): e1009256.