Fig. 3: SYCP2 plays an essential role in transcription-coupled homologous recombination (TC-HR).

A Scheme of tet-DR-GFP HR reporter for measuring TC-HR efficiency was shown on top. Relative HR frequency was measured when the transcription is on or off by qRT-PCR in siCtrl or siSYCP2 treated cells. Mean frequency of the HR (qPCR) compared to the siCtrl group at transcription off is shown. Three independent experiments were done (n = 3 experiments, Mean +/− SEM). (p = 0.0002). B Scheme of damage induction via KillerRed (KR) at transcribed or non-transcribed region of the genome in the DART assay (left). U2OS-TRE cells were transfected with GFP-SYCP2 and TA-KR/tetRKR/TA-cherry/tetR-cherry. For light activation of KR in all below experiments in the DART assay, cells were light-activated for 20 min and recovered for 30 min. γH2AX foci were stained and are positive at sites of KR but not cherry. SYCP2 is preferentially recruited to sites of TA-KR. Representative images and quantification of recruitment of SYCP2 at the indicated site were shown. Mean intensity of SYCP2 at TA-KR /mean intensity of background was shown (n = 10 cells, Mean +/− SEM). Experiments were repeated 3 times. C SYCP2 foci frequency at I-SCEI endonuclease-induced damage sites marked by TA-Cherry in U2OS-TRE cells treated 24 h with or without I-SCEI transfection. Three independent experiments were done (n = 3 experiments, Mean +/− SEM). D U2OS-TRE cells transfected with TA-KR and siCtrl/siSYCP2 were light-activated, recovered for 30 min, fixed, and stained with anti-RAD51. Fold increase of RAD51 foci at sites of KR compared to background was quantified (n = 25 cells for siCtrl and n = 16 cells for siSYCP2, Mean +/− SEM). Experiments were repeated 3 times. E U2OS-TRE cells transfected with TA-KR and siCtrl/siSYCP2 with or without light-activation were recovered at 4 h and 24 h, then fixed and stained with anti-S9.6. Frequency of S9.6 foci positive cells at TA-KR was counted. Three experiments were done (n = 3 experiments, Mean +/− SEM), 100–200 individual cells were quantified per group. Statistical analysis was done with the unpaired two-tailed Student-t-test, ****p < 0.0001. Scale Bar = 10 μm. Source data are provided as a Source Data file.