Fig 2: TIRR binds RNA with hairpin and single-stranded structures(A) Principles of the surface plasmon resonance (SPR) assay. His-TIRR is immobilized on a sensor chip surface, increasing concentrations of RNA and DNA substrates are added, and binding is measured. Image was created with BioRender.com.(B) SPR with His-TIRR and (left ? right) ssRNA, RNA hairpin, RNA beacon, dsRNA, ssDNA, DNA hairpin, dsDNA, and RNA:DNA hybrid. Increasing concentrations of DNA/RNA added up to 200 µM.(C) Summary of (B). Table showing the calculated binding constant (Kd) from the SPR data.(D) Quantification of EMSAs with ssRNA (n = 4), RNA beacon (n = 3), and ssDNA (n = 3) and increasing concentrations of TIRR.(E) Quantification of EMSAs showing comparison with ssRNA and increasing concentration of TIRR (as shown in D) and 53BP1-Tudor (n = 3) (mean ± SD).(F) Quantification of EMSAs showing comparison with RNA beacon and increasing concentration of TIRR (as shown in D) and 53BP1-Tudor (n = 3) (mean ± SD).(G) Quantification of EMSAs showing comparison with ssDNA and increasing concentration of TIRR (as shown in D) and 53BP1-Tudor (n = 2) (mean ± SD).

Fig 3: Dicer and Drosha, but not RNA:DNA hybrids, are required for TIRR/53BP1 separation(A) PLA of TIRR and 53BP1 with and without IR (10 Gy, 1 h) with transfection prior to damage of either mock, WT RNasH1, or WKKD mutant RNaseH1. n(mock - IR) = 329, n(mock + IR) = 487, n(WT - IR) = 135, n(WT + IR) = 98, n(WKKD - IR) = 102, n(WKKD + IR) = 89.(B) Quantification of (A) (mean ± SD, n = 2).(C) Western blot showing induction of ?H2AX after IR and expression of RNaseH1 WT and WKKD mutant.(D) Immunofluorescence microscopy showing induction of ?H2AX after IR and nuclear localization of RNaseH1.(E) PLA of TIRR and 53BP1 with and without IR (10 Gy, 1 h) with knockdown of Dicer or Drosha prior to damage induction. n(siNeg - IR) = 551, n(siNeg + IR) = 428, n(siDicer - IR) = 459, n(siDicer + IR) = 391, n(siDrosha - IR) = 336, n(siDrosha + IR) = 271.(F) Quantification of (E) (mean ± SD, n = 2).(G) Western blot showing knockdown of Dicer and Drosha and induction of ?H2AX after IR.(H) Scheme depicting the involvement of Dicer and Drosha in the RNA-dependent DNA-damage response and miRNA pathways, and DGCR8 involvement in miRNA processing. Image was created with BioRender.com.(I) PLA of TIRR and 53BP1 with and without IR (10 Gy, 1 h) with knockdown of DGCR8 prior to damage induction. n(siNeg - IR) = 114, n(siNeg + IR) = 178, n(siDGCR8 - IR) = 155, n(siDGCR8 + IR) = 251.(J) Quantification of (I) (mean ± SD, n = 2).(K) Western blot of DGCR8 knockdown.

Fig 4: Hairpin RNA promotes dissociation of the TIRR/53BP1 complex in vitro and in vivo(A) Principles of the homogeneous time-resolved fluorescence (HTRF) assay with TIRR and 53BP1. Image was created with BioRender.com.(B) Graph showing data from the HTRF assay with increasing concentrations of ssRNA 1, ssRNA 2, dsRNA, RNA hairpin, RNA/DNA hybrid 1, and RNA/DNA hybrid 2. (mean ± SD, n = 6).(C) PLA of TIRR and 53BP1 1 or 3 h after transfection of a Cy3-labeled RNA hairpin (100 nM). n(mock) = 275, n(1 h) = 266, n(3 h) = 315.(D) Quantification of (C) (mean ± SD, n = 2).(E) PLA of TIRR and Cy3 1 or 3 h after transfection of a Cy3-labeled RNA hairpin (100 nM). n(mock) = 313, n(1 h) = 241, n(3 h) = 291.(F) Quantification of (E) (mean ± SD, n = 2).

Fig 5: The TIRR/53BP1 complex is localized to DSBs prior to dissociation(A) PLA of TIRR and ?H2AX, 53BP1 and ?H2AX, or ?H2AX alone with and without IR (10 Gy, 1 h) (n(?H2AX - IR) = 85, n(?H2AX + IR) = 75, n(?H2AX + TIRR - IR) = 73, n(?H2AX + TIRR + IR) = 105, n(?H2AX + 53BP1 - IR) = 123, n(?H2AX + 53BP1 + IR) = 122).(B) Quantification of (A) (mean ± SD, n = 2).(C) PLA of TIRR and ?H2AX with and without IR (10 Gy, 1 h) in the presence of a-Amanitin (2 µg/mL, 24 h) or DRB (100 µM, 1 h). n(DMSO - IR) = 291, n(DMSO + IR) = 357, n(AA - IR) = 303, n(AA + IR) = 303, n(DRB - IR) = 348, n(DRB + IR) = 383.(D) Quantification of (C) (mean ± SD, n = 2).(E) Co-immunoprecipitation of TIRR-GFP or GFP with and without IR (10 Gy, 1 h). Ponceau is used as a loading control.(F) Quantification of (E), ?H2AX bound to TIRR-GFP ± IR (mean ± SD, n = 3).(G) Quantification of (E), 53BP1 bound to TIRR-GFP ± IR (mean ± SD, n = 3).(H) Laser stripping of 53BP1-GFP U2OS cells and TIRR-GFP HeLa cells (mean ± SD, n = 1).(I) PLA of TIRR and ?H2AX with and without IR (10 Gy, 1 h) with transfection prior to damage of either mock, WT Neongreen-TIRR, or K10E Neongreen-TIRR. n(mock - IR) = 347, n(mock + IR) = 402, n(WT - IR) = 329, n(WT + IR) = 339, n(K10E - IR) = 432, n(K10E + IR) = 338.(J) Quantification of (I) (mean ± SD, n = 2).(K) PLA of Neongreen and ?H2AX with and without IR (10 Gy, 1 h) with transfection prior to damage of either mock, WT Neongreen-TIRR, or K10E Neongreen-TIRR. n(mock - IR) = 315, n(mock + IR) = 309, n(WT - IR) = 343, n(WT + IR) = 289, n(K10E - IR) = 324, n(K10E + IR) = 258.(L) Quantification of (K) (mean ± SD, n = 2).