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. 2021 Jun 23;10:e69454. doi: 10.7554/eLife.69454

Figure 3. EXOSC1 cleaves ssDNA.

(A) Coomassie blue staining of in vitro purified EXOSC1 protein. (B) Schematic of synthetic DNA substrates. (C) In vitro cleavage assays of EXOSC1 using generic ssDNA, dsDNA, and DNA-RNA hybrid as substrates. The assays were performed in the presence of 1 μM oligonucleotides, 1 μM EXOSC1, 70 mM KCl, 700 μM MgCl2, 1 mM DTT, and 20 mM Tris–HCl pH 7.0. After incubation at 37°C for 4 hr, resultant samples were analyzed by 15% polyacrylamide TBE-urea gels. (D) Cleavage assays of EXOSC1 in the presence or absence of EXOSC3, EXOSC6, EXOSC7, and EXOSC8 using generic ssDNA as substrates. (E) Cleavage assays in the presence of the components as noted. (F) Cleavage assays of EXOSC1 at the pH as noted. (G) Time course cleavage assays of EXOSC1 using generic ssDNA as substrates. (H) Rate curve of EXOSC1 cleavage at 37°C and pH 7.0. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

Figure 3.

Figure 3—figure supplement 1. EXOSC1 cleaves ssDNA.

Figure 3—figure supplement 1.

(A) In vitro cleavage assays of EXOSC1 homologs (EXOSC2–EXOSC9) using generic ssDNA, dsDNA, and DNA-RNA hybrid as substrates. (B) Cleavage assays of EXOSC1 in the presence/absence of EXOSC2, EXOSC4, EXOSC5, or EXOSC9. (C) Cleavage assays of exosome complex (EXOSC1–9) using generic ssDNA. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.