Fig 1: Tyrosine 65 (Y65) of RAD52 is required for the recruitment and the interaction with POLD3. (A) The recruitment of POLD3 to KR-TRF1 in U2OS RAD52 KO cells after transient expressing RAD52 mutants. (B) Co-immunoprecipitation of GFP-RAD52 WT and mutants with POLD3 after KR-TRF1-induced damage for 1 h (left). Co-immunoprecipitation of GFP-RAD52 WT with POLD3 after treating cell lysates with ethidium bromide (0.1 mg/ml) or RNase H (20 U/ml) for 30 min (right). (C) The clearance of γ-H2AX after POLD3 knockdown by siRNA. (D) Colony formation assays for U2OS and U2OS POLD3 knockdown by siRNA and transiently expressing KR-TRF1 or RFP-TRF1 with indicated time to light exposure. n = 3, error bars represent SD. (E) Schematic model of R-loop–CSB–RAD52 mediates BIR contribute to the repair of telomeric DSB induced by ROS. The current model is composed of the following sequential steps: ROS-induced R-loop formation is dependent on TERRA and TRF2; CSB–ADC–R464 recognizes R-loop; K141/144 mediates R-loop binding of RAD52; RAD52 R65 mediated POLD3 interaction promotes BIR. For (a) and (c), Mean values with SD from 50 cells in three independent experiments are given. P-value is calculated by unpaired t-test. ***P < 0.001.
Fig 2: RAD52 is recruited to telomeric R-loop via CSB and K144-mediated hybrid binding. (A) The recruitment of RAD52 to damaged telomere in U2OS CSB KO cells after light exposure for 30 min and 60 min. (B) Schematic model of RAD52 structure and mutants. Right panel is Monomer structure of RAD52 and mutant sites. Y65A (orange), K141A (yellow), K144A (blue), R153A (purple), R156A (pink). (C) The recruitment of RAD52 mutants to KR-TRF1 after ROS damage. Quantification of the percentage of RAD52 co-localization with KR-TRF1. (D) DNA: RNA hybrids EMSA assay of purified RAD52 WT and RAD52 K144A protein. For (A) and (C), mean values with SD from 50 cells in three independent experiments are given. P-value is calculated by unpaired t-test. ***P < 0.001.
Fig 3: CSB is recruited to telomeric R-loops and contributes to cell survival and telomeric integrity. (A) Colony formation assays for U2OS and U2OS CSB KO cells with transiently expressing KR-TRF1 or RFP-TRF1. n = 3, error bars represent SD. (B) Telomere aberrations were found in U2OS and U2OS CSB KO cells with or without transiently expressing KR-TRF1. (C) Recruitment of CSB in KR-TRF1 or RFP-TRF1 expressing U2OS cells with or without light activation. Quantification of co-location frequency of myc-CSB and KR-TRF1/RFP-TRF1. (D) CSB foci co-localization with KR-TRF1 in U2OS cells treated with KU55933 (ATM inhibitor, 10 uM, 24 h), Nu7026 (DNA-PK inhibitor, 20 uM, 24 h), Olaparib (10 uM, 24 h), XAV-933(Tankyrase inhibitor,20 uM, 24 h), ATR inhibitor (10 uM, 24 h), and CDK2 inhibitor (5 uM, 24 h). (E) The staining of CSB at KR-TRF1 in U2OS cells after treating with overexpressed with HA-RNaseH WT, D210N mutant, DRB (20 μM, 24 h) and α-amanitin (100 μg/ml, 2 h), or TERRA knockdown by LNA. (F) CSB foci co-localization with KR-TRF1 in U2OS cells treated with TRF2 knockdown. (G) The staining of S9.6 at KR-TRF1 in U2OS cells after treating with CSB knockdown by siRNA. For (D)–(H), mean values with SD from 50 cells in three independent experiments are given. P-value is calculated by unpaired t-test, **P < 0.01, ***P < 0.001.
Fig 4: CSB modulates SNM1A exonuclease activity. Single-stranded or double-stranded 3′-end labeled 61-mer (A) or 21-mer (B) oligonucleotides were incubated with SNM1A (+; fixed concentration of 0.8 nM), and increasing concentrations of CSB where indicated (0.8–50 nM), for 30 min at 37°C. Reactions were resolved on a denaturing gel, and images were captured during phosphorimager analysis (shown is a representative gel from three independent experiments). Note that the non-labeled strand of the double-stranded substrate is blocked at its 5′-end with a biotin moiety (B) to prevent competing digestion of this strand.
Fig 5: CSB-RAD52 cascade contributes to the repair of ROS induced telomeric DSB. (A) Colony formation assays for U2OS CSB KO cells with RAD52 knockdown by siRNA transiently expressing KR-TRF1. (B) The intensity of GFP-XRCC1 foci after CSB or RAD52 knockdown by siRNA at 4h and 24h after light activation. (C) The clearance of γ-H2AX after RAD52 knockdown. (D) The recruitment of RAD52 to RFP/KR/FOK1-TRF1after light activation or transfection. (E) The recruitment of RAD52 to KR-TRF1 after treating with DRB (20 μM, 24 h) and α-amanitin (100 μg/ml, 2 h), TERRA knockdown by LNA, or overexpressed with HA-RNaseH WT, D210N mutant. (F) Telomere aberrations were found in U2OS and U2OS RAD52 KO cells with or without transiently expressing KR-TRF1. For (B), (C), (D) and (E), Mean values with SD from 50 cells in three independent experiments are given. P-value is calculated by unpaired t-test. ***P < 0.001.
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