Nucleolar protein nucleolin functions in replication stress-induced DNA damage responses

Abstract

The nucleolus contains multiple copies of ribosomal (r)DNA, which indicate sites of frequent replication stress and suggest the existence of a mechanism to prevent replication stress-related rDNA instability and the possibility that such a mechanism contributes to the whole genomic stability against replication stress. We have previously reported that nucleolin, a major nucleolar protein, is involved in ionizing radiation-induced DNA damage responses (DDRs) such as ataxia telangiectasia mutated (ATM)-dependent cell cycle checkpoints and homologous recombination (HR) repair. Here, we investigated the role of nucleolin in DDR due to replication stress. The results indicate that following replication stress, nucleolin interacted with the histone γH2AX, proliferating cell nuclear antigen (PCNA), and replication protein A (RPA)32, suggesting that it may be recruited to DNA damage sites on the replication fork. Furthermore, the knockdown of nucleolin by siRNA reduced the activation of ATM and RAD3-related (ATR) kinase and the formation of RAD51 and RPA32 foci after replication stress due to UV or camptothecin exposure, whereas nucleolin overexpression augmented ATR-dependent phosphorylation and RAD51 and RPA accumulation on chromatin. Moreover, these overexpressing cells seemed to increase repair activity and resistance to replication stress. Our results indicate that nucleolin plays an important role in replication stress-induced DDRs such as ATR activation and HR repair. Given that nucleolin overexpression is often observed in many types of cancer cells, our findings suggest that nucleolin is involved in the regulation of resistance to replication stress that may otherwise lead to tumorigenesis and it could be a possible target for chemotherapy and radiotherapy.

Keywords: ATR; DNA damage; nucleolin; nucleolus; replication stress.

Fig. 1.

Nucleolin (NCL) participates in replication stress–induced responses. (A) GFP-FBL–expressing U2OS cells were irradiated by γ-rays (5 Gy: γH2AX and 53BP1; 10 Gy: RAD51) following incubation for 30 min (γH2AX and 53BP1) or 4 h (RAD51), or treated with CPT (0.5 μM, 1.5 h: γH2AX and 53BP1; 2 μM, 2 h: RAD51). Then, their cells were fixed after the incubation time for IR or completion of CPT treatment, and immunostaining was performed using the indicated antibodies and visualized to the red color with the secondary antibody. The green color is derived from GFP-FBL. (B) GFP-FBL–expressing U2OS cells were transfected by nucleolin siRNA or negative control siRNA, and after 2 days these cells were fixed and immunostaining was performed using anti-γ-H2AX antibody. (C and D) Nucleolin interacts with γH2AX and PCNA following replication stress. Extracts from HeLa cells, treated with 10 Gy of γ-rays or 2 μM of CPT, were immunoprecipitated with anti-γH2AX antibody (C), anti-PCNA antibody (D), and then the immunocomplexes were detected by western blot analysis using the indicated antibodies.

Fig. 2.

Nucleolin (NCL) contributes to ATR-dependent phosphorylation following replication stress. U2OS cells were transfected by nucelolin siRNA. After 2 days, these cells were irradiated by UV (A: 10 J; C: 5 J) or treated with 2 μM of CPT (B). Then, their cells were harvested at the indicated times and analyzed by western blot using the indicated antibodies. Arrows indicate the bands of mono-ubiquitinated PCNA in (A) and (B). Quantification of pATR of Fig. 2C and the other two experiments were carried out by ImageJ software and are shown in the lower panel of Fig. 2C (*P < 0.05).

Fig. 3.

Nucleolin (NCL) participates in the HR pathway following replication stress. (A and B) U2OS cells were transfected by nucleolin siRNA or negative control siRNA, and after 2 days these cells were treated with 2 μM of CPT. After 4 h, their cells were fixed and immunostaining was performed using the anti-RAD51 antibody (A) or the anti-RPA32 antibody (B). Then, percentages of foci-positive cells were counted under the fluorescence microscope (*P < 0.01; **P < 0.02). (C) Nucleolin is indispensable for the chromatin accumulation of HR factors. U2OS cells were transfected by nucleolin siRNA or negative control siRNA, and after 2 days these cells were treated with 2 μM of CPT. After the indicated times, their cells were harvested and their chromatin fractions were prepared. Chromatin accumulation of DDR proteins were detected by western blot analysis using the indicated antibodies.

Fig. 4.

Overexpression of nucleolin (NCL) amplified DNA damage responses with replication stress. (A and B) Nucleolin interacts with RPA and Rad17. Extracts from HeLa cells, treated with 10 Gy of γ-rays or 2 μM of CPT, were immunoprecipitated with anti-RPA32 antibody (A), or anti-Rad17 antibody (B), and then the immunocomplexes were detected by western blot analysis using the indicated antibodies. (C) Nucleolin-overexpressing or mock cells were irradiated by 5J of UV. Then, their cells were harvested at the indicated times and analyzed by western blot using the indicated antibodies. (D) Nucleolin-overexpressing (NCL#5) or mock cells were treated with 2 μM of CPT. After the indicated times, their cells were harvested and their chromatin fractions were prepared. Chromatin accumulation of DDR proteins were detected by western blot analysis using the indicated antibodies.