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Abstract

Multiple DNA repair pathways may be involved in the removal of the same DNA lesion caused by endogenous or exogenous agents. Although distinct DNA repair machinery fulfill overlapping roles in the repair of DNA lesions, the mechanisms coordinating different pathways have not been investigated in detail. Here, we show that Ku70, a core protein of nonhomologous end-joining (NHEJ) repair pathway, can directly interact with DNA polymerase-β (Pol-β), a central player in the DNA base excision repair (BER), and this physical complex not only promotes the polymerase activity of Pol-β and BER efficiency but also enhances the classic NHEJ repair. Moreover, we find that DNA damages caused by methyl methanesulfonate (MMS) or etoposide promote the formation of Ku70-Pol-β complexes at the repair foci. Furthermore, suppression of endogenous Ku70 expression by small interfering RNA reduces BER efficiency and leads to higher sensitivity to MMS and accumulation of the DNA strand breaks. Similarly, Pol-β knockdown impairs total-NHEJ capacity but only has a slight influence on alternative NHEJ. These results suggest that Pol-β and Ku70 coordinate 2-way crosstalk between the BER and NHEJ pathways.-Xia, W., Ci, S., Li, M., Wang, M., Dianov, G. L., Ma, Z., Li, L., Hua, K., Alagamuthu, K. K., Qing, L., Luo, L., Edick, A. M., Liu, L., Hu, Z., He, L., Pan, F., Guo, Z. Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-β and Ku70.

Keywords: DNA repair; base excision repair; double-strand break.

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Conflict of interest statement

The authors thank Dr. Songbai Liu (Suzhou Vocational Health College, Suzhou, China) for providing the U2OS EJ5-GFP cell line, and Dr. Jun Huang (Zhejiang University, Hangzhou, China) for providing the U2OS alt-NHEJ-EGFP cell line, which were very helpful for testing NHEJ efficiency in vivo. This work was supported by the National Natural Science Foundation of China (81872284), the Changzhou Science and Technology Program (CE20175035), the Jiangsu Key Research and Development Program (Grant BE2018714), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (18KJA180006), the National Nature Science Foundation (31701179), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. G.L.D. was funded by the Russian Foundation for Basic Research (RFBR) according to Research Project N°19-04-00067. The authors declare no conflicts of interest.

Figures

Figure 1

Figure 1

Ku70 interacts with Pol-β. A …

Figure 1

Ku70 interacts with Pol-β. A ) Cell lysates were immunoprecipitated with antibodies against…

Figure 1 Ku70 interacts with Pol-β. A) Cell lysates were immunoprecipitated with antibodies against Ku70 or normal IgG. Associated proteins were analyzed by Western blot with indicated antibodies. B, C) Purified Ku70 and Pol-β were immunoprecipitated with indicated antibodies or normal IgG (as a control) and analyzed by Western blot.
Figure 2

Figure 2

The Ku70 and Pol-β interaction…

Figure 2

The Ku70 and Pol-β interaction is enhanced in response to DNA damage. A …

Figure 2 The Ku70 and Pol-β interaction is enhanced in response to DNA damage. A) HEK293T cells stably expressing Flag-Pol-β were treated with 1 mM MMS or PBS for 30 min and left to recover for 1 h. Cells were lysed, and proteins were immunoprecipitated from the lysates using anti-Flag M2 Beads. The precipitates were then subjected to Western blot analysis. B) Similar to A, HEK293T cells stably expressing Flag-Pol-β were treated with 10 μM ETO or DMSO for 1.5 h and released for 1 h. Cell lysates were prepared and subjected to immunoprecipitation and Western blot analysis with the indicated antibodies. The numbers in the figure represent the relative gray values of the bands below (regard control treatment group as 1, quantified with ImageJ software). C, D) HEK293T cells were treated with 1 mM MMS (C, the bottom part of the panel) and 10 μM ETO (D, lower panel) or treated with control reagents (C, D, upper panel) for 30 min and left to recover for 1 h. Ku70 and Pol-β were detected using antibodies specific for Ku70 and Pol-β, which were visualized with Alexa Fluor 488– or 594–conjugated secondary antibodies, respectively, followed by confocal microscopy. Colocalization was visualized by using the ImageJ colocalization finder plugin. Colocalization of Ku70 (green) and Pol-β (red) in cells appears yellow in merged images. Quantification of the colocalization between Ku70 and Pol-β was performed by using the colocalization function of ImageJ from 150 cells. This experiment was repeated 3 times. Scale bars, 10 μm. ***P < 0.001 (2-sided Student’s t test).
Figure 3

Figure 3

Ku70 promotes BER capacity by…

Figure 3

Ku70 promotes BER capacity by stimulating Pol-β activity. A ) SP-BER was reconstituted…

Figure 3 Ku70 promotes BER capacity by stimulating Pol-β activity. A) SP-BER was reconstituted with purified recombinant proteins and FAM-labeled U-containing oligonucleotide duplex substrate. Ku70 protein (0, 50, 100 ng, 0.8 μg) or similar amount of BSA were added to reconstituted reactions. B) LP-BER was reconstituted with purified recombinant proteins and FAM-labeled F-containing oligonucleotide duplex substrate. Ku70 protein (0, 50, 100 ng, 0.8 μg) or similar amount of BSA were added to the reconstituted reactions. C) Polymerase activity assay was performed using 20 ng purified Pol-β and FAM-labeled oligonucleotide duplex substrate. Where indicated, Ku70 protein (0, 50, 100 ng, 0.8 μg) or similar amount of BSA were added. At the top part of each panel, there is a schematic presentation of the substrate used in the experiments. The bottom part of panels A and B, and right part of panelC show quantification of the corresponding experiments. F, tetrahydrofuran; U, uracil. Values represent the mean ± sd of 3 independent experiments. **P < 0.01, ***P < 0.001 (2-sided Student’s t test).
Figure 4

Figure 4

Ku70 knockdown reduces BER efficiency. …

Figure 4

Ku70 knockdown reduces BER efficiency. A , B ) SP-BER and LP-BER were…

Figure 4 Ku70 knockdown reduces BER efficiency. A, B) SP-BER and LP-BER were carried out with different concentrations of extracts prepared from Ku70-depleted cells or control cells as described in Materials and Methods. The top part of each panel shows the schematic structure of the corresponding DNA substrate. The bottom charts are a quantification of 3 independent repeats of the experiments for A and B, respectively. ***P < 0.001 (2-sided Student’s t test).
Figure 5

Figure 5

Suppressing Ku70 sensitizes cells to…

Figure 5

Suppressing Ku70 sensitizes cells to MMS and induces more DNA single-strand breaks. A …

Figure 5 Suppressing Ku70 sensitizes cells to MMS and induces more DNA single-strand breaks. A) HEK293T cells transfected with Ku70 siRNA or scramble siRNA were captured under the microscope after 6-h treatment with 1 mM MMS. Scale bar, 30 μm. B) Relative cell viability was examined after 12-h treatment with 1 mM MMS. Statistical analysis of 3 independent experiments was accomplished as indicated. C, D) HEK293T cells transfected with Ku70 siRNA or scramble siRNA were treated with 1 mM MMS for 30 min, and percentage of DNA in the tails was quantified by the Comet assay immediately after treatment. Scale bar, 10 μm. Data represent the mean ± sd of 3 independent experiments. **P < 0.01, ***P < 0.01 (2-sided Student’s t test).
Figure 6

Figure 6

Pol-β involves in NHEJ-mediated DNA…

Figure 6

Pol-β involves in NHEJ-mediated DNA repair. A , B ) The schematic representation…

Figure 6 Pol-β involves in NHEJ-mediated DNA repair. A, B) The schematic representation of EJ5-GFP (A) and alt-NHEJ-GFP (B) reporter system. C) U2OS EJ5-GFP cells were transfected with Pol-β shRNA or control shRNA for 24 h, and then cells were transfected with HA-I-SceI expression plasmid and continuously cultured for 48 h. Cells were collected for FACS analysis to identify GFP-positive cells. D) alt-NHEJ-EGFP U2OS cells were transfected with Pol-β shRNA or control shRNA for 24 h and then transfected with the HA-I-SceI expression plasmid and further incubated for 48 h; cells were then collected for FACS analysis. Knockdown efficiency and transfection efficiency were confirmed by Western blot as shown in the bottom part of panels C and D). NS, no significance. Data represent the mean ± sd of 3 independent experiments. **P < 0.01 (Student’s t test). E) Colocalization of Pol-β and γ-H2AX in DMSO or 10 μM ETO-treated HEK293T cells. Representative images are shown. Pol-β (green), γ-H2AX (red), or merged images are indicated. Scale bar, 10 μm.
Figure 7

Figure 7

Pol-β–deficient cells exhibit more sensitive…

Figure 7

Pol-β–deficient cells exhibit more sensitive to ETO and delay DSB repair. A )…

Figure 7 Pol-β–deficient cells exhibit more sensitive to ETO and delay DSB repair. A) Representative Western blot. Scramble shRNA and Pol-β shRNA–transfected HeLa cells were treated with DMSO or 10 μM ETO for 1.5 h and harvested at the indicated time points after treatment. Cells were lysed, and Western blot analysis was performed using specific antibodies against Pol-β, Ku70, γ-H2AX, Ku80, and Tubulin. B) Densitometric quantification of γ-H2AX from 3 independent experiments was performed with ImageJ software. C) Scramble shRNA and Pol-β shRNA–transfected HeLa cells were treated same as in A, and then fixed at the indicated times. Cells were stained with an anti–γ-H2AX antibody, and nucleus was stained using DAPI and then visualized by a fluorescence microscope. D) Densitometric quantification of γ-H2AX. Data represent the mean ± sd of 100 cells from 3 independent experiments. E) HeLa cells transfected with scramble shRNA or Pol-β shRNA were treated with indicated concentrations of ETO, and cell survival was analyzed. Data represent the mean ± sd of 3 independent experiments. Scale bar, 10 μm. **P < 0.01, ***P < 0.001 (2-sided Student’s t test).
Figure 8

Figure 8

Graphical summary of the model…

Figure 8

Graphical summary of the model for BER-NHEJ crosstalk mediated by Ku70 and Pol-β…

Figure 8 Graphical summary of the model for BER-NHEJ crosstalk mediated by Ku70 and Pol-β supported by this study. BER is the major SSB repair pathway, and c-NHEJ is the major DSB repair pathway. When SSBs occur, Ku70 binds to Pol-β and promotes polymerase activity of Pol-β, thus accelerating BER. Reciprocally, when DSBs occur, Pol-β binds to Ku70 and participates in c-NHEJ to promote repair of the DSB lesions.
All figures (8) See this image and copyright information in PMC

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