DNA damage repair elements accumulate at DNA damage sites to form ionizing radiation-induced foci (IRIF) for damage repair. IRIF, which represent direct evidence of DNA damage response activity, which are conveniently to be observed via immunofluorescence staining. Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, the substrate binding protein E3 ubiquitin-ligases enzymes are recruited to DNA damage sites, then the E2 ubiquitin-conjugating enzymes are recruited to these sites by the E3 where they catalyze protein ubiquitination. However, IRIF of E2 enzymes are relatively transient and unstable in vivo and difficult to detect. Here, we present a new method for the observation of E2 IRIF. This method is based on the co-transfection of interacting E2 and E3 enzymes into cells and identifies IRIF via immunofluorescence following DNA damage.
Keywords: DNA damage; Foci; Ubiquitin; Ubiquitin-conjugating enzyme.