Extended Data Fig. 6: RNA-DNA hybrids contribute to DNA damage in micronuclei.
From: Breakage of cytoplasmic chromosomes by pathological DNA base excision repair
(a) Dot blot showing that the α-inosine antibody recognizes dI in dsDNA oligonucleotides. α-dsDNA is the loading control. (b) dI accumulates on MN in MPG KO cells. Aggregated data from four biological replicates to detect α-inosine signal from purified PN and MN from wild-type and two MPG KO clones of U2OS cells. (mean with SD, Ordinary one-way ANOVA with Dunnett’s multiple comparisons test, *P = 0.0465 (KO1), *P = 0.0212(KO2)). (c) Aggregated data from three replicate experiments for the example in Fig. 3c and 3d. Inosine+ ruptured MN were defined as having >3SD of the PN mean value (mean with SD, ****P < 0.0001, two-tailed unpaired t-test. control: n = 577; MPG KO: n = 882). (d) Overexpression of ITPA does not reduce DNA damage in MN. MN γ-H2AX FI measured from control or cells overexpressing ITPA. ITPA expression was induced with CMV promoter (mean with 95% CI, NS, P = 0.3736, Two-tailed Mann-Whitney U test, control: n = 129; ITPA: n = 124). (e) Aggregated data from three replicate experiments to detect MN DNA damage after ITPA overexpression as in Extended Data Fig. 6d (mean with SD, NS, P = 0.2306, two-tailed unpaired t-test. control: n = 274; ITPA: n = 372). (f) Data from four independent replicates of the experiment in Fig. 3e, showing S9.6 enrichment in MN over PN. Y axis shows S9.6 labeling intensity for PN and MN fractions from four biological replicates (mean with SD, **P = 0.0020, unpaired t test after log transformation). (g) Western blot to detect induced expression of RNaseH1 (Tet-on system). GAPDH is the loading control. (h) RNaseH1 over expression suppresses MN DNA damage. Aggregated data from three replicate experiments for the example shown in Fig. 3g (mean with SD, NS, P = 0.9355, ***P = 0.0001, Ordinary one-way ANOVA with Dunnett’s multiple comparisons test. control: n = 399; RNaseH1: n = 391; RNaseH1-CI (D210N): n = 548). (i) Overexpression of RNaseH1 suppresses MN DNA damage in U2OS cells. MN γ-H2AX FI from cells with or without overexpression of RNaseH1 or a catalytically inactive mutant (D210N) in U2OS cells (mean with 95% CI, ****P < 0.0001, Ordinary one-way ANOVA with Dunnett’s multiple comparisons test. control: 141; RNASEH1: n = 168; RNASEH1-CI: n = 125). (j) Overexpression of RNaseH1 suppresses MN DNA damage in U2OS cells. Summary of three biological replicates as in Extended Data Fig. 3i above (mean with SD, NS, P = 0.3963, *** P = 0.0002, Ordinary one-way ANOVA with Dunnett’s multiple comparisons test. control: n = 548; RNASEH1: n = 470; RNASEH1-CI: n = 380). (k) Western blot to detect induced expression of the C-terminal catalytic domain of SETX (C-SETX, Tet-on system). GAPDH is the loading control. (l) C-SETX overexpression suppresses MN DNA damage. MN γ-H2AX FI in cells with or without overexpression of C-SETX (mean with 95% CI, ****P < 0.0001, Two-tailed Mann-Whitney U test. control: n = 149; C-SETX: n = 118). (m) Aggregated data from three replicates for overexpression of C-SETX as in Extended Data Fig. 3l (mean with SD, ****P < 0.0001, two-tailed unpaired t-test. control: n = 392; C-SETX: n = 579).
