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. Author manuscript; available in PMC: 2022 Jul 19.
Published in final edited form as: Nature. 2021 Oct 20;599(7884):308–314. doi: 10.1038/s41586-021-04009-w

Fig. 3. Apoptotic DNA fragmentation and subsequent ligation by Lig3 is required for eccDNA production in mESCs.

Fig. 3.

a-b, EccDNA production is induced by apoptosis. mESCs were treated with the indicated apoptosis inducers, and total DNA (a) and eccDNAs (b) were purified. DMSO: Dimethylsulfoxide; STS: Staurosporine; ETO: etoposide; UV: ultraviolet.

c-d, Deficiency of oligonucleosomal DNA fragmentation abolishes apoptosis induced eccDNA production. Deficiency of DNase 𝛾−/−, but not EndoG−/−, abolishes UV-induced oligonucleosomal DNA fragmentation (c), and eccDNA production (d) in mESCs. MtDNA was kept as an inner control.

e, Confirmation of DNA ligases deficient cell lines. Up, immunoblotting confirming knockout of DNA ligases in CH12F3 cell lines. Bottom, genomic structure of Lig3 with CRISPR/Cas9 specific targeting (Δ) NucLig3 but retaining MtLig3 for cell viability.

f-g, Lig3 is the major DNA ligase for eccDNA generation. Staurosporine induced oligonucleosomal DNA fragmentation (f) and eccDNA (g) from the indicated CH12F3 cell lines. Amount of input cells, elution and loading volume were equal among samples within each agarose gel (a-d, and f-g). Shown are representatives of three independent experiments.