Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-based Reporter Assays

J Vis Exp. 2024 Jun 14:(208). doi: 10.3791/66969.

Abstract

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli. Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence. These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Publication types

  • Video-Audio Media

MeSH terms

  • DNA Breaks, Double-Stranded*
  • DNA Repair* / physiology
  • Genes, Reporter
  • High-Throughput Screening Assays / methods
  • Humans
  • Luciferases / genetics
  • Luciferases / metabolism
  • Luminescent Measurements / methods

Substances

  • Luciferases