Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin

Abstract

Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (∼ 90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.

Figure 1.

Chromatin status at site-specific genomic DNA by targeting tetR-KR or TA-KR to an integrated TRE site. (A) Scheme of tetR- and TA-tagged KR expression in the U2OS TRE cell line. To induce ROS-mediated damage at a specific locus in the genome, we fused KR to the tetracycline repressor (tetR) to induce ROS damage in a 90-kb TRE array (total of 96 repeats) in U2OS cells. In the cell line, 96 random TRE repeats were integrated into the genome site in U2OS cells at ∼200 copies. The plasmid also contains 24 tandem MS2 bacteriophage viral replicase translational operators (MS2 repeats) and SKL-tagged CFP after the TRE array and CMV promoter. (B) FISH assay of the U2OS TRE cell line. Blue is DAPI staining. Red is staining of the TRE region. Green is the X-chromosome specific probe. (C) Incorporation of EU, translocation of GFP-MS2 and expression of SKL-CFP were monitored in U2OS TRE cell line 24 h after transfection with tetR or TA tagged mcherry. (D) U2OS TRE cells transfected with either tetR or TA were stained with phospho-RNA Pol II antibody 4H8, which specifically recognizes pSer5 of the CTD heptapeptide of RNA Pol II. Quantification of localization of 4H8 staining and recruitment of YFP Pol II at the site of tetR and TA–KR in U2OS TRE cells transfected with tetR-KR or TA-KR is shown. (E) Localization of GFP-HP1α in U2OS TRE cells transfected with either tetR-cherry or TA-cherry (upper panel). U2OS TRE cells transfected with either tetR-KR or TA-KR were fixed and stained with H3AcK9 (lower panel). (F) Right panel shows the magnification of colocalization of HP1α and H3AcK9 at the site of tetR or TA. The intensity of HP1α and AcK9H3 at the site of tetR and TA was quantified. Mean value with SD is the intensity in 10 cells.

Figure 2.

Site-specific genomic DNA damage by targeting tetR-KR or TA-KR to an integrated TRE site. (A and B) U2OS TRE cells transfected with tetR or TA–KR with or without exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 min were stained after treatment with anti-PAR (A), anti-8-oxoG (B) and anti-KR (−Light: without light activation, +Light: with light activation). (C and D) Graph shows the percentage of colocalization between PAR, oxoG and KR in 50 cells. Mean values with a SD in three independent experiments are given in all of the following quantification graphs. The P-value is calculated by Student’s t-test using stat plus software; P < 0.005 is shown as double asterisks. (E) U2OS TRE cells transfected with tetR-KR were treated with NAC (20 mM) or MnTBAP (100 µM) immediately before exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 min. Cells were fixed and stained with anti-8-oxoG after light activation. The intensity of 8-oxoG (average of 10 cells) at the site of tetR was quantified. SD is the intensity of 8-oxoG in 10 cells. (F) U2OS TRE cells transfected with tetR or TA–KR with or without exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 min were stained after treatment with anti-γH2AX and KR (−Light: without light activation, +Light: with light activation). (G) U2OS TRE cells were treated with NAC (20 mM) and MnTBAP (100 µM) immediately before exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 min. Cells were fixed and stained with anti-γH2AX after light activation. The intensity of γH2AX at the site of tetR was quantified. Mean value with SD is the intensity of 8-oxoG in 10 cells.

Figure 3.

KR activation with either light or 559-nm laser excitation efficiently induces recruitment of DNA glycosylases. (A) Damage response of NTH1 to the site of tetR-KR (left) or TA-KR (right)-induced DNA damage 3 min after 559 nm laser (50 mW) bleaching. The red rectangle shows the selected bleaching area of the 559-nm laser. (B) Quantification of kinetics of GFP-NTH1 (green) and tetR-KR (red) at the site of damage. The mean intensity of each data point was obtained after subtraction of the background intensity in the irradiated cell. (C) Quantification of damage response of GFP-NTH1 3 min after the indicated laser light activation of tetR-KR. The fold increase was obtained from the foci intensity/the background intensity at the site of KR. Data are mean values with a SD of three cells. (D) Damage response of NEIL1 and NEIL2 to the site of tetR (left) or TA (right)—KR-induced DNA damage with the exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 min. (E) Quantification of percentage of cells with colocalization of GFP-NEIL1 or NEIL2 at the site of tetR or TA before and after a 15-W SYLVANIA cool white fluorescent bulb for 10 min. (F) U2OS TRE cells were treated with NAC (20 mM) and MnTBAP (100 µM) immediately before exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 min. Cell were fixed in 5-min light activation. The relative intensity of NTH1 at the site of tetR (intensity of NTH1 at the site of tetR/intensity of NTH1 area away from tetR) was quantified. Mean value with SD is the relative intensity of NTH1 in 10 cells.

Figure 4.

Comparison of the recruitment of repair factors to KR plus light-induced damage and 405-nm laser-induced damage. (A) GFP-NTH1 and tetR-KR were expressed in the U2OS TRE cell line. KR was bleached at full power 559-nm laser light for 50 scans with a rate of 1 mW/scan (total 50 mW); another indicated point in the same cell was irradiated with full power 405-nm laser light for 10 scans with a rate of 5 mW/scan (total 50 mW). (B) The quantification of damage response of GFP-NTH1 at the site of KR-induced damage or 405-nm laser-induced damage 3 min after irradiation. The fold increase was obtained from foci intensity/background intensity. Mean values with a SD of 10 cells are given. (C and D) The damage response of GFP-XRCC1 under the same conditions as in (A) and quantification of the damage response of GFP-XRCC1 at the site of KR-induced damage or 405-nm laser-induced damage 3 min after irradiation. (E) Schematic of deletion mutants of XRCC1 used in this study. (F) Quantification of the damage response ability of domains of XRCC1 relevant to full-length XRCC1. The average mean intensity of each domain (BRCT I, LI360/361DD, 1-300NTD, BRCT II)/mean intensity of full-length XRCC1 in response to laser-induced SSBs (gray) or KR induced base damage (black) is shown. Mean values with a SD of three independent experiments.

Figure 5.

KR activation induces recruitment of DNA Polß at the sites of both tetR and TA in U2OS TRE and U2OS 263 cell lines. (A) Damage response of Polß to the site of tetR or TA-fused mCherry or KR 3 min after 559-nm laser light irradiation in U2OS TRE cell line. (B) Graph shows the percentage of colocalization between Polß and tetR and TA-fused mCherry (left), Polß and tetR or TA-fused KR (right) in 50 cells in U2OS TRE cell line. Mean values with a SD of three independent experiments. (C) The recruitment of Polß at the site of tetR-KR in U2OS (263) cell line 3 min after 559-nm laser bleaching.

Figure 6.

PARP1 is efficiently recruited to the site of tetR-KR but not TA-KR, while FEN1 is recruited to the site of TA-KR but not tetR-KR. (A) The damage response of PARP1 (upper panel) or FEN1 (lower panel) in U2OS TRE cells at the site of tetR-KR (left) or TA-KR (right)-induced damage is shown. (B and C) Quantification of the damage response of GFP-PARP1 (B) and FEN1 (C) at the site of tetR-KR or TA-KR induced damage in U2OS TRE cell line. The fold increase was obtained from foci intensity/background intensity. Mean values with a SD of 10 cells are given in (B), (C) and (D). (D) Quantification of the damage response of GFP-PARP1 at the site of tetR-KR or TA-KR induced damage in the U2OS 263 cell line. (E) The GFP-XRCC1 or Polß and tetR-KR expressing-U2OS TRE cell line was treated with 10 µM olaparib in medium for 30 min. The damage response of GFP-XRCC1 or Polß was measured 3 min after 559-nm laser bleaching at the site of tetR-KR. The suppression effects of olaparib on the damage response of GFP-XRCC1 and Polß are shown by comparison to the mean intensity of full-length protein w/o olaparib treatment. (F) The damage response of FEN1 to TA-KR-induced damage 3 min after 559-nm laser bleaching with treatment of 10 µM olaparib in medium for 30 min. Mean values with a SD in 10 cells are given.

Figure 7.

The recruitment of FEN1 to TA-KR-induced damage is dependent on PCNA. (A) The damage response of domains and mutations of FEN1 to TA-KR-induced damage 3 min after 559 nm laser bleaching. The N-terminus of FEN1 (N-ter), D184A (flap endonuclease mutant), C-terminus of FEN1 (C-ter) and FF343/344AA (PCNA-binding motif mutation) were used. (B) The damage response of GFP-PCNA to tetR- or TA-KR-induced damage 3 min after 559-nm laser bleaching. (C) U2OS TRE cells expressing myc-cdt1, GFP-PARP1 and tetR-KR (left) or TA-KR (right). Damage response of PARP1 to the site of tetR (left) or TA (right)—KR-induced DNA damage with exposure to a 15 W SYLVANIA cool white fluorescent bulb for 10 min in cdt1 expressing cells (upper panel) or not expressing cells (lower panel) is shown. (D) U2OS TRE cells expressing myc-cdt1, GFP-FEN1 or PCNA and TA-KR. Damage response of FEN1 and PCNA at the site of TA-KR-induced DNA damage with exposure to a 15 -W SYLVANIA cool white fluorescent bulb for 10 min in cdt1-expressing cells (upper panel) or not expressing cells (lower panel) is shown. (E) U2OS TRE cells expressing GFP-FEN1, GFP-PCNA and TA-KR are pretreated with or without the RNA polymerase II inhibitor 20 µM DRB in medium for 24 h. The damage response of PCNA and FEN1 at the site of TA-KR-induced DNA damage with exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 min is shown. (F) U2OS TRE cells transfected with sicontrol, siFEN1, siFEN1+ WT-FEN1 or siFEN1+FF343/344AA. A total of 5000 cells were seeded into 96-well plates; 48 h after preparation, cells were treated with H₂O₂ in PBC for 1 h at the indicated concentration for the MTT assay. Mean values with a SD for three experiments are given. (G) A model of response of repair factors to oxidative DNA damage at condensed chromatin or open chromatin.