TdT-dUTP DSB End Labeling (TUDEL), for Specific, Direct In Situ Labeling of DNA Double Strand Breaks.

Abstract

The genome of a living cell is continuously damaged by various exogenous and endogenous factors yielding multiple types of DNA damage including base damage and damage to the sugar-phosphate backbone of DNA. Double Strand Breaks (DSBs) are the most severe form of DNA damage and if left unchecked, may precipitate genomic rearrangements, cell death or contribute to malignancy. In clinical contexts, radiation is often used to induce DSBs as a form of genotoxic therapy. Despite the importance of DSBsand their repair, as yetthere is no facile assay to detect DSBs in situ or to quantify their location or proximity to other cellular constituents. Such an assay would help to disentangle DDR signaling pathways and identify new molecular players involved in DSB repair. These efforts, in turn, may facilitate drug screening and accelerate the discovery of novel, more effective genotoxic agents. We have developed such an assay, presented here, and term it TdT-dUTP DSB End Labeling (TUDEL).TUDEL makes use of Terminal Deoxynucleotidyl Transferase (TdT), a template-independent DNA polymerase. TdT is commonly used in TUNEL assays to yielda binary output of DNA damage. We haveadapted this approach, usingTdT and EdUTP tolabel individual DNA double strand breaks in irradiated cellsand detecting the incorporated EdU with fluorescent probes via Click chemistry.This tool complements and is compatible with existing, indirect methods to track DSBssuch as immunofluorescent detection of γH2AX. TUDEL is also sufficientlyspecific, sensitive,quantitative,and robustto replace the neutral Comet assay for routine measurement of DSB formation and repair. Here we present a protocol for TUDEL.