HR/NHEJ GFP Reporter Assay

Overview

The HR/NHEJ GFP reporter assay system uses stably integrated fluorescent reporter constructs to quantify the efficiency of DNA double-strand break (DSB) repair pathways in live cells. The DR-GFP (direct repeat GFP) construct measures homologous recombination (HR) efficiency; EJ5-GFP measures non-homologous end joining (NHEJ); an MMEJ-GFP construct measures microhomology-mediated end joining (MMEJ). A DSB is induced by I-SceI endonuclease expression, and the fraction of GFP-positive cells quantified by flow cytometry reports pathway activity.

Used by

  • DR-GFP (HR), EJ5-GFP (NHEJ), and MMEJ-GFP reporter assays used in U2OS cells to demonstrate that TRMT10A knockdown reduces HR efficiency and increases compensatory NHEJ and MMEJ, whereas TRMT10A catalytic-dead mutant G206R fully rescued HR; ATM-phospho-deficient S28A mutant failed to rescue HR PMID:28068672.

Notes

  • Assays performed in U2OS cells (standard DDR reporter host) for mechanistic studies.
  • GFP-positive fraction measured by flow cytometry to quantify pathway activity.
  • Used alongside immunofluorescence foci assays (γ-H2AX, BRCA1, RAD51) for orthogonal validation.

Sources

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