WRN participates in translesion synthesis pathway through interaction with NBS1

Abstract

Werner syndrome (WS), caused by mutation of the WRN gene, is an autosomal recessive disorder associated with premature aging and predisposition to cancer. WRN belongs to the RecQ DNA helicase family, members of which play a role in maintaining genomic stability. Here, we demonstrate that WRN rapidly forms discrete nuclear foci in an NBS1-dependent manner following DNA damage. NBS1 physically interacts with WRN through its FHA domain, which interaction is important for the phosphorylation of WRN. WRN subsequently forms DNA damage-dependent foci during the S phase, but not in the G1 phase. WS cells exhibit an increase in spontaneous focus formation of poleta and Rad18, which are important for translesion synthesis (TLS). WRN also interacts with PCNA in the absence of DNA damage, but DNA damage induces the dissociation of PCNA from WRN, leading to the ubiquitination of PCNA, which is essential for TLS. This dissociation correlates with ATM/NBS1-dependent degradation of WRN. Moreover, WS cells show constitutive ubiquitination of PCNA and interaction between PCNA and Rad18 E3 ligase in the absence of DNA damage. Taken together, these results indicate that WRN participates in the TLS pathway to prevent genomic instability in an ATM/NBS1-dependent manner.

Figure 1

NBS1-dependent WRN focus formation in response to DNA damage. (A) Formation of WRN foci in response to several DNA-damaging agents. HeLa cells were treated by several DNA-damaging agents as indicated. After 4 hours, cells were fixed and immuno-staining was performed using anti-WRN antibody. (B) Formation of WRN foci in NBS cells after DNA damage. Normal (NBS1-complemented) and NBS cells were treated by IR (5 Gy of γ-ray). After 1 hour, their cells were fixed and immuno-staining was performed using anti-WRN antibody. (C) Physical interaction of WRN with NBS1. Extracts from normal and NBS lymphoblastoid cells, harvested at 30 minutes after 10 Gy of IR, were immunoprecipitated with anti-WRN antibody and the immuno-complexes was detected by Western blot analysis. (D) Focus formation of several DNA damage response factors in WS cells. WS cells were treated by IR (5 Gy of γ-ray). After 30 minutes, their cells were fixed and immuno-staining was performed.

Figure 2

NBS1 interacts with WRN and contributes to ATM-dependent WRN phosphorylation. (A) ATM/NBS1-dependent phosphorylation of WRN. Normal (MRC5), NBS and AT fibroblast cells were irradiated with 5 Gy of γ-rays. These cells were harvested at the indicated times after IR and analyzed by Western blot using anti-WRN antibody. Phosphorylated WRN was detected as the slower-migrating band. (B) Schematics showing protein structure and design constructs of NBS1. MBD means hMre11-binding domain and AIM means ATM-interacting motif. NBS1-R2 mutant truncates C-terminus (670aa-754aa), FHA-2D mutant substitutes both Gly27 and Arg28 to Asp and BRCT-2D mutant substitute both Lys150 and Val 151 to Asp. These mutated NBS1 were generated and used in (Sakamoto et al., 2007; Tauchi et al., 2001). (C) Interaction of NBS1 with WRN through the FHA domain. NBS cells, expressing each mutated NBS1 construct, was harvested at 30 minutes after 10 Gy of IR and then were immunoprecipitated with anti-NBS1 antibody and the immuno-complexes was detected by Western blot analysis. (D) WRN phsophorylation was investigated in the NBS cells, expressing FHA-truncating NBS1 or wild type NBS1 (Full) by Western blot analysis. (E) Formation of WRN foci in ATM-deficient cells. AT or HeLa cells were treated by CPT (1 μM). After 1 hour, their cells were fixed and immuno-staining was performed using anti-WRN antibody. +caffeine: pre-treatment by 1 mM caffeine.

Figure 3

WRN responds to S phase-dependent DNA damage. (A) IR-induced activation of ATM-related pathway. WS and normal cells were irradiated by 5 Gy of γ-ray. These cells were harvested at 0.5 hour after IR and analyzed by Western blot using indicated antibodies. (B) S phase-dependent WRN focus formation. Normal primary fibroblasts (TIG-3) were incubated in low serum media for 48 hours and then stimulated to proliferate by exchanging with high serum media (Fukami et al., 1995). After then, these cells were treated by etoposide (30 μM) for 4 hours and immuno-staining was performed using anti-WRN and anti-phospho-DNA-PKcs (T2609) antibodies. G1: at 2 hours after serum stimulation. S: at 12 hours after serum stimulation. (C) Formation of polη foci in WRN-knockdown cells. GFP-polη-expressing HeLa cells were transfected by WRN siRNA. After 2 days, their cells were treated by UV (20 J/m²). After 4 hour, their cells were fixed and the focus formation of GFP-polη-was observed and counted under a fluorescent microscope. (D) Formation of Rad18 foci in WS cells after CPT treatment. Normal (48BR) and WS cells were treated by CPT (2 μM). After 4 hour, their cells were fixed and immuno-staining was performed using anti-Rad18 antibody and Rad18 foci-positive cells were counted under a fluorescent microscope. (E) supF mutant frequency in WS cells. UV-irradiated or un-irradiated pSP189 was transfected into normal (48BR) and WS cells. After 48 hours, replicated pSP189 was recovered from cells, and supF mutant frequencies were determined as described in Material and Methods.

Figure 4

WRN interacts with PCNA and might contribute to translesion synthesis. (A)(B) Physical interaction of WRN with PCNA. The extracts from normal or NBS lymphoblastoid cells, harvested at indicated times after 10 Gy of IR (A) at 1 hour after 1 μM of CPT treatment (B), were immunoprecipitated with anti-WRN antibody or normal rabbit IgG, and then the immuno-complexes was detected by Western blot analysis. Ratios about immunoprecipitated PCNA were calculated to each un-treated sample by ImageJ software. (C) DNA damage-dependent interaction between PCNA and Rad18. The extracts from normal (48BR) or WS cells, harvested at 1 hour after 1 μM of CPT treatment, were immunoprecipitated with anti-Rad18 antibody, and then the immuno-complexes was detected by Western blot analysis. Ratios of immunoprecipitated PCNA were calculated to un-irradiated sample (normal cells) by ImageJ software. (D) Spontaneous ubiquitination of PCNA in WS cells. Normal (48BR) and WS cells were treated by 5 Gy of γ-ray or 1 μM of CPT. These cells were harvested at the indicated times after DNA damage and analyzed by Western blot using anti-PCNA antibody. Ubiquitinated PCNA was detected as the delayed band (Ub). Ratios of ubiquitinated PCNA were calculated to un-irradiated sample (normal cells) by ImageJ software. (E)(F) DNA damage-dependent degradation of WRN. Normal, AT, and NBS lymphoblastoid cells (E) or 48BR cells (F) were treated by 1 μM of CPT. These cells were harvested at the indicated times after CPT treatment and analyzed by Western blot using anti-WRN antibody. Ratios were calculated to each un-irradiated sample by ImageJ software. Arrow: WRN protein, *: non-specific bands. ATM inhibitor: pre-treatment of KU-55933 [10 μM, 1 hour] (Hickson et al., 2004).

Figure 5

WRN participates in the regulation of PCNA mono-ubiquitination and subsequent TLS through interaction with PCNA. WRN is phosphorylated by ATM/NBS1 in response to DNA damage and this phosphorylation leads to a degradation of WRN. PCNA is released from WRN complex following WRN degradation and then could be mono-ubiquitinated by Rad18/Rad6. Mono-ubiquitination of PCNA activates translesion DNA synthesis.