Timely termination of repair DNA synthesis by ATAD5 is important in oxidative DNA damage-induced single-strand break repair

Abstract

Reactive oxygen species (ROS) generate oxidized bases and single-strand breaks (SSBs), which are fixed by base excision repair (BER) and SSB repair (SSBR), respectively. Although excision and repair of damaged bases have been extensively studied, the function of the sliding clamp, proliferating cell nuclear antigen (PCNA), including loading/unloading, remains unclear. We report that, in addition to PCNA loading by replication factor complex C (RFC), timely PCNA unloading by the ATPase family AAA domain-containing protein 5 (ATAD5)-RFC-like complex is important for the repair of ROS-induced SSBs. We found that PCNA was loaded at hydrogen peroxide (H2O2)-generated direct SSBs after the 3'-terminus was converted to the hydroxyl moiety by end-processing enzymes. However, PCNA loading rarely occurred during BER of oxidized or alkylated bases. ATAD5-depleted cells were sensitive to acute H2O2 treatment but not methyl methanesulfonate treatment. Unexpectedly, when PCNA remained on DNA as a result of ATAD5 depletion, H2O2-induced repair DNA synthesis increased in cancerous and normal cells. Based on higher H2O2-induced DNA breakage and SSBR protein enrichment by ATAD5 depletion, we propose that extended repair DNA synthesis increases the likelihood of DNA polymerase stalling, shown by increased PCNA monoubiquitination, and consequently, harmful nick structures are more frequent.

Figure 1.

ATAD5 accumulates on chromatin upon oxidative DNA damage. (A) The scheme for generation of a HeLa cell line expressing mNeonGreen-mAID-tagged ATAD5 (HeLa-ATAD5^{mNeonGreen-AID} cell). (B) HeLa-ATAD5^{mNeonGreen-AID} cells were treated with auxin for 6 h and fixed for immunostaining. (C) HeLa-ATAD5^{mNeonGreen-AID} cells were treated with auxin for indicated times, and then detergent-insoluble proteins were fractionated and subjected to immunoblotting. (D and E) HeLa-ATAD5^{mNeonGreen-AID} cells were treated with 10 mM H₂O₂ for 10 min (D) or with various DNA-damaging agents as indicated (E) and fixed after pre-extraction with CSK buffer (upper panel in D and E) or without pre-extraction (lower panel in D). (D) The fixed cells were subjected to immunostaining with an anti-XRCC1 antibody. Four-fold magnified images are shown in the corner. (F) HeLa-ATAD5^{mNeonGreen-AID} cells were incubated with EdU for 1 h before detergent-pre-extraction. Cells were treated with H₂O₂ as indicated, detergent-pre-extracted and fixed for EdU-click reactions. mNeonGreen signals were quantified in strong-EdU-signal-negative cells. Red bar indicates mean value. Statistical analysis: two-tailed unpaired Student's t-test; ****P < 0.001. (G) HeLa-ATAD5^{mNeonGreen-AID} cells were subjected to 405-nm UV laser microirradiation. Two minutes after microirradiation, cells were fixed for immunostaining as indicated. (B and G) Scale bar: 10 μm. (D and E) Scale bar: 20 μm.

Figure 2.

ATAD5 accumulation upon oxidative DNA damage depends on RFC1. (A–C) HeLa-ATAD5^{mNeonGreen-AID} cells transfected with small interfering RNAs (siRNAs) as indicated for 48 h. Cells were treated with 10 mM H₂O₂ for 10 min, detergent-pre-extracted and then fixed for detection of mNeonGreen signal. (A) Representative images are shown; scale bar: 20 μm. (B) Quantification of mNeonGreen signals. Three independent experiments were performed and one representative result is displayed. Red bar indicates mean value. (C) Results of immunoblotting of whole cell extracts prepared 48 h after transfection. (D and E) HeLa cells were treated with nocodazole for 5 h, shaked-off and released into fresh medium followed by incubation for 4 h before treatment with DNA-damaging agents under conditions used in Figure 1D and E. After damage treatment, cells were, pre-extracted with detergent before fixation and immunostained with an anti-PCNA antibody. (D) Representative images are shown; scale bar: 10 μm. (E) The intensity of PCNA signal was quantified. PCNA intensity of four independent experiments are normalized. Error bars represent standard deviation of the mean (n = 4). (F) U2OS-ATAD5^AID cells previously reported (14) were enriched at G1 phase by treatment with PD 0332991 and PHA-767491, and treated with DNA-damaging agents under conditions used in Figure 1D and E. After drug treatment, detergent-soluble and -insoluble proteins were fractionated and subjected to immunoblotting. (B and E) Statistical analysis: two-tailed unpaired (B) and paired (E) Student's t-test; ***P < 0.005, *P < 0.05, and ns: not significant.

Figure 3.

Accumulation of PCNA and ATAD5 upon oxidative DNA damage depends on 3′-terminal-processing enzymes. (A–M) HeLa-ATAD5^{mNeonGreen-AID} cells were transfected with siRNAs as indicated and incubated for 48 h before 10 mM H₂O₂ treatment for 10 min. (A,B,D,E,G and H) Cells were incubated with EdU for 30 min before detergent-pre-extraction. (J and K) Before H₂O₂ treatment, cells were treated with nocodazole for 5 h, shaked-off and released into fresh medium followed by incubation for 4 h. (A, B, D, E, G, H, J and K) After H₂O₂ treatment, cells were detergent-pre-extracted and fixed for EdU-click reactions (A, B, D, E, G, H) and PCNA immunostaining. PCNA (A, D, G, J) and mNeonGreen (B, E, H, K) signal intensity was quantified. (A, B, D, E, G, H) Quantification was performed in strong-EdU-signal-negative cells. Three independent experiments were performed and one representative result is displayed. Red bar indicates mean value. Statistical analysis: two-tailed unpaired Student's t-test; ****P < 0.001. (C) Forty-eight hours after transfection, protein was extracted and subjected to immunoblotting. (F, I, L, M) Forty-eight hours after transfection, RNA was extracted and subjected to reverse transcription-quantitative PCR (RT-qPCR) with the specific primer set for each gene.

Figure 4.

H₂O₂ sensitivity and single-strand DNA breakage are increased in ATAD5-depleted cells. (A) U2OS-ATAD5^AID cells pre-treated with auxin were treated with H₂O₂ for 4 h with MTT reagents, and cell survival was measured by quantification of MTT formazan. Error bars represent standard deviation of the mean (n = 3). (B–E) U2OS-ATAD5^AID cells treated with auxin for 6 h (B and C) and U2OS cells (D) or HeLa cells (E) transfected with ATAD5 siRNA for 48 h were treated with 0.1 mM H₂O₂ for 1 h and collected for an alkaline COMET assay. (B) Representative images of an alkaline COMET assay. (C–E) The tail moment was calculated from ∼200 cells and plotted. Three independent experiments were performed and one representative result is displayed. Red bar indicates mean value. Statistical analysis: two-tailed unpaired Student's t-test; ****P < 0.001, *P < 0.05. (F and G) HeLa-ATAD5^AID cells (F) and U2OS-ATAD5^AID cells (G) were pre-treated with auxin for 24 and 48 h, respectively, and fixed to prepare metaphase spreads. Sister chromatid exchange (SCE) events were counted in metaphase spreads. Statistical analysis: two-tailed unpaired Student's t-test; **P < 0.01, *P < 0.05. (H) U2OS-ATAD5^AID cells pre-treated with auxin were treated methyl methanesulfonate (MMS) for 1 h, and cell survival was measured by cellular ATP quantitation. Error bars represent standard deviation of the mean (n = 3). (D and E) 3′UTR: siRNA targeting 3′-untranlsated regions (UTR) of ATAD5 gene, #3: siRNA #3 targeting an exon region of ATAD5 gene.

Figure 5.

PCNA accumulates on DNA upon oxidative damage in ATAD5-depleted cells at G1 phase. (A) HeLa-ATAD5^{mNeonGreen-AID} cells were transfected with ATAD5 siRNA targeting 3′UTR and subjected to 405-nm UV laser microirradiation. Two minutes after microirradiation, cells were fixed for PCNA immunostaining; scale bar: 10 μm. (B–F) Asynchronous U2OS-ATAD5^AID cells (B and C) or U2OS-ATAD5^AID cells G1-enriched by treatment with PD 0332991 and PHA-767491 (D–F) were treated with auxin for 16 h and then treated with H₂O₂ as indicated. (B–E) After H₂O₂ treatment, cells were detergent-pre-extracted, fixed and immunostained with anti-PCNA antibody. (B and D) Representative images of PCNA immunostaining; scale bar: 20 μm. (C and E) Quantification of chromatin-bound PCNA signal intensity. Three independent experiments were performed and one representative result is displayed. Red bar indicates mean value. Statistical analysis: two-tailed unpaired Student’s t-test; ****P < 0.001. (G and H) U2OS cells (G) or HeLa cells (H) transfected with ATAD5 siRNAs were enriched at G1 phase by treatment with PD 0332991 and PHA-767491 (G) or by releasing cells from nocodazole arrest (H), and then treated with 0.5 mM H₂O₂ for 1 h. #1 and #3: siRNAs targeting an exon region of ATAD5 gene. (F–H) After H₂O₂ treatment, detergent-insoluble proteins were isolated and subjected to immunoblotting.

Figure 6.

Unscheduled DNA synthesis (UDS) is increased in ATAD5-depleted cells. (A–G) U2OS-ATAD5^AID cells treated with auxin (A–D) and MRC-5 cells transfected with ATAD5 siRNA targeting 3′UTR (E–G) were enriched at the G1 phase by releasing cells from nocodazole arrest; cells were treated with 0.5 mM H₂O₂ for 1 h or 1 mM H₂O₂ for 20 min with EdU incorporation, detergent-pre-extracted, and fixed for EdU-click reaction and immunostaining. UDS was measured based on EdU signal incorporation. (A and E) Representative images of immunostained cells treated with 1 mM H₂O₂ for 20 min are shown; scale bar: 10 μm. (E) The dotted line represents the edge of the nucleus. (B–D, F, G) The mean signal intensity of EdU (B and F), PCNA (C and G) and Pol δ (D) was quantified. (H) HeLa-ATAD5^{mNeonGreen-AID} cells were transfected with ATAD5 siRNA targeting 3′UTR and then subjected to 405-nm UV laser microirradiation. Two minutes after microirradiation, cells were fixed and immunostained as indicated; scale bar: 10 μm. (I and J) U2OS-ATAD5^AID cells enriched at G1 phase by treatment with PD 0332991 and PHA-767491 were treated with auxin for 12 h before H₂O₂ treatment. Cells were then detergent-pre-extracted and fixed for immunostaining of XRCC1 (I) or PAR (J). Cells were incubated with EdU for 30 min before detergent-pre-extraction. (K and L) U2OS-ATAD5^AID cells were treated with auxin for 12 h, irradiated with 10 J/m² UV-C, incubated with EdU for 3 h, and subjected to PCNA immunostaining (K) and UDS assay (L). Intensity of PCNA (K) and EdU (L) signals was quantified. (B–D, F, G, I–L) Three independent experiments were performed and one representative result is displayed. Red bar indicates mean value. Statistical analysis: two-tailed unpaired Student's t-test; ****P < 0.001, **P < 0.01.

Figure 7.

PCNA-unloading-defective ATAD5 fails to restore defects in ATAD5-depleted cells. (A–D) U2OS-ATAD5^AID cells were transfected with cDNA expressing mNeonGreen protein-tagged wild-type (WT) ATAD5 or PCNA-unloading-defective ATAD5 E1173K (EK) mutant for 48 h before 1 mM H₂O₂ treatment for 20 min with EdU incorporation. Auxin was added 12 h before H₂O₂ treatment. Cells were then detergent-pre-extracted and fixed for immunostaining and UDS assay. (A) Representative images of cells treated with auxin and H₂O₂. The dotted line represents the edge of the nucleus; scale bar: 10 μm. (B–D) mNeonGreen (B), PCNA (C) and EdU signals (D) were quantified and displayed; Ne: negative, Po: positive. (E and F) U2OS cells were transfected with a combination of WT or E1173K mutant ATAD5 (EK) cDNA and ATAD5 siRNA targeting 3′UTR for 48 h. (E) Detergent-insoluble proteins were fractionated and immunoblotted. (F) Cells were treated with 0.1 mM H₂O₂ for 1 h and subjected to an alkaline COMET assay. (B–D) Three independent experiments were performed and one representative result is displayed. Red bar indicates mean value. (F) Six independent experiments were normalized. Error bars represent standard deviation of the mean. (B–D, F) Statistical analysis: two-tailed unpaired (B–D) and paired (F) Student's t-test (F); ****P < 0.001, **P < 0.01, *P < 0.05 and ns: not significant. (G) Graphical model for extended DNA synthesis and frequent exposure of nicks in ATAD5-depleted cells. (a) In wild-type cells treated with H₂O₂, (a, i) when Pol δ encounters DNA lesion on template DNA during repair DNA synthesis, it stalls and leaves from PCNA, and PCNA is monoubiquitinated. (a, ii) TLS polymerases are recruited and bypass the DNA lesion. (a, iii,iv) PCNA-Pol δ then takes over DNA synthesis until unloaded by ATAD5-RLC. After PCNA unloading, nucleosome is assembled. The remaining DNA lesions on template DNA are removed by another round of repair mechanism. (b, c) In ATAD5-depleted cells, repair DNA synthesis is extended due to the less nucleosome compaction occurred locally around clustered DNA damages (b, i) or globally (c) by PCNA remaining on DNA (red), or other yet-clear mechanism, (b, ii and c) which increases the likelihood that Pol δ will encounter DNA lesions and expose nicks. (b, iii and c) Pol δ leaves PCNA and DNA synthesis is terminated.