Nonenzymatic role for WRN in preserving nascent DNA strands after replication stress

Abstract

WRN, the protein defective in Werner syndrome (WS), is a multifunctional nuclease involved in DNA damage repair, replication, and genome stability maintenance. It was assumed that the nuclease activities of WRN were critical for these functions. Here, we report a nonenzymatic role for WRN in preserving nascent DNA strands following replication stress. We found that lack of WRN led to shortening of nascent DNA strands after replication stress. Furthermore, we discovered that the exonuclease activity of MRE11 was responsible for the shortening of newly replicated DNA in the absence of WRN. Mechanistically, the N-terminal FHA domain of NBS1 recruits WRN to replication-associated DNA double-stranded breaks to stabilize Rad51 and to limit the nuclease activity of its C-terminal binding partner MRE11. Thus, this previously unrecognized nonenzymatic function of WRN in the stabilization of nascent DNA strands sheds light on the molecular reason for the origin of genome instability in WS individuals.

Figure 1. WRN stabilizes nascent DNA strands in response to replication stress

(A) Collapsed replication forks are shortened in cells derived from WS patients. Replicating DNA in hTERT-immortalized WS and WS cells complemented with wild-type WRN (WS+WRN) were first labeled with IdU for 30 min and then treated with or without CPT (1 μm) for 5 hours. DNA fibers were immunostained with anti-BrdU antibodies. DNA fiber images were captured using a fluorescence microscopy and DNA fiber lengths were measured using Axiovision Software. The frequency distributions of lengths of more than 100 DNA fibers from three or four independent experiments in each group were calculated. Inset shows western blot for WRN expression in WS and WS+WRN cells. See also Figure S1, Tables S1 and S2. (B) Nascent DNA tracts are shortened in the absence of WRN in HeLa cells exposed to CPT. HeLa cells were transfected with either WRN or control shRNA, labeled with IdU for 30 min, treated with CPT or mock-treated for 5 h, and the frequency distributions of lengths of more than 100 DNA fibers from three independent experiments in each group were calculated. Inset shows western blot for WRN expression in HeLa cells transfected with WRN shRNA and control shRNA. (C) Nascent DNA strands are shortened in WRN-deficient mouse embryonic fibroblasts (MEFs) treated with CPT. MEFs derived from WRN-defective and wild-type mice were labeled with IdU for 30 min, treated with 1 μm CPT or mock-treated for 5 h, and the frequency distributions of lengths of more than 100 DNA fibers from three independent experiments in each group were calculated. (D) Replication forks are stable in WS cells in response to low doses of CPT. WS and WS+WRN cells were labeled with IdU for 30 min, treated with 25 or 100 nM CPT for 5 h, and the frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. (E) Replication forks are stable in WS cells in response to short HU treatment. WS and WS+WRN cells were labeled with IdU for 30 min, treated with 4 mM HU for 5 h, and the frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. (F) Replication forks are shortened in BRCA2-deficient V-C8 cells in response to short HU treatment. V-C8 and V-C8+BRCA2 cells were labeled with IdU for 30 min, treated with 4 mM HU for 5 h, and the frequency distributions of lengths of DNA fibers were calculated.

Figure 2. WRN preserves nascent DNA strands in response to replication breaks

(A) Replication forks are shortened in WS cells in response to long HU treatment. WS and WS+WRN cells were labeled with IdU for 30 min, treated with 4 mM HU for 24 h, and the frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. (B) WRN stabilizes nascent DNA strands in response to replication-associated DSBs. WS and WS+WRN cells were labeled with IdU for 30 min and then exposed to 2 μm aphidicolin/1 μm CPT or CPT only for 5 h. The frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated.

Figure 3. N-terminal region of WRN (1-366 amino acids) is sufficient to stabilize nascent DNA strands in response to replication-associated DSBs

(A) Schematics show different functional regions of human and mouse WRN and the various WRN domains used in the study. TDD: WRN-WRN interaction domain; HRDC: helicase and RNase D C-terminal domain; RQC: RecQ conserved domain: NLS: nucleolar localization signal; E84A: exonuclease mutant; K577A: helicase mutant. See also Figure S3. (B) N-terminal 1-366 aa domain, exonuclease-defective (E84A), and helicase-defective (K577A) WRN form foci in response to replication stress. WS cells stably expressing WRN_1-366, WRN_250-366, WRN_940-1432, WRN_E84A and WRN_K577A were treated with 1 μM CPT for 1 h. After 5 h, WS cells expressing WRN_1-366, WRN_250-366 and WRN_940-1432 were immunostained with anti-γH2AX, and WS cells expressing WRN_E84A and WRN_K577A were immunostained with anti-γH2AX and anti-WRN antibodies. Representative confocal microscope images are shown. Scale bars, 5 and 10 μm. DAPI, 4′6-diamidino-2-phenylindole. (C) WRN_1-366 is involved in the maintenance of nascent DNA tracts. SV40-immortalized WS cells stably expressing WRN_1-366 were labeled with IdU for 30 min, treated with 1 μM CPT or mock-treated for 5 h and the frequency distributions of lengths of more than 100 DNA fibers from three independent experiments in each group were calculated. (D) WRN-WRN interaction domain (WRN_250-366) is not sufficient to stabilize nascent DNA tracts. SV40-immortalized WS cells stably expressing WRN_250-366 were labeled with IdU for 30 min, treated with 1 μm CPT or mock-treated for 5 h, and the frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. (E) The C-terminal region containing HRDC and DNA binding motifs (940-1432 aa) of WRN is not involved in the maintenance of nascent DNA strands. SV40-immortalized WS cells stably expressing WRN_940-1432 were labeled with IdU for 30 min, treated with 1 μM CPT or mock-treated for 5 h, and the frequency distributions of lengths of more than 100 DNA fibers from three independent experiments in each group was calculated. (F) The helicase domain of WRN is not required for the maintenance of nascent DNA strands. MEFs derived from helicase domain deficient WRN (WRN^Δhel/Δhel) mice were labeled with IdU for 30 min, treated with 1 μM CPT or mock-treated for 5 h, and the frequency distributions of lengths of more than 100 DNA fibers from three independent experiments in each group were calculated. (G-H) The replication fork maintenance function of WRN is independent of its exonuclease and helicase activities. WS cells stably expressing exonuclease-defective (E84A, G) or helicase-defective (K577A, H) WRN were labeled with IdU for 30 min, treated with 1 μM CPT or mock-treated for 5 h, and the frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. Inset shows western blot analyses for WT, E84A and K577A WRN expression in WS cells.

Figure 4. Nascent DNA strand maintenance function of WRN is NBS1 dependent

(A) Diagram shows different functional protein-protein interaction domains of NBS1 protein. FHA: Forkhead-associated domain; BRCT: BRCA1 C-terminus domain; WRN, ATM and MRE11: WRN, ATM and MRE11 interaction domains, respectively. (B) FHA domain of NBS1 is essential for the recruitment of WRN to the sites of replication-associated DSBs. Representative confocal images show recruitment of WRN to the sites of replication-associated DSBs in NBS cells expressing full-length NBS1. NBS, NBS+NBS1, and NBS+ΔFHA-NBS1 cells were treated with 1 μM or were mock-treated for 5 h and then immunostained with anti-WRN and anti-γH2AX antibodies. Scale bars, 5 μm. DAPI, 4′6-diamidino-2-phenylindole. (C) Nascent DNA strands are intact in NBS cells complemented with NBS1. NBS+NBS1 cells were labeled with IdU for 30 min, exposed to 1 μm CPT or were mock-treated for 5 h. Frequency distributions of lengths of more than 100 DNA fibers from three independent experiments in each group were calculated. (D) FHA domain of NBS1 is required for the maintenance of nascent DNA strands after replication breaks. NBS cells stably expressing ΔFHA NBS1 were labeled with IdU for 30 min, exposed to 1 μm CPT or were mock-treated for 5 h, and frequency distributions of lengths of more than 100 DNA fibers from three independent experiments in each group were calculated. Inset shows western blot analysis for NBS1 and ΔFHA-NBS1 expression in NBS cells.

Figure 5. MRE11 degrades nascent DNA strands in response to collapsed replication forks in the absence of WRN

(A) Nascent DNA tract lengths are maintained in NBS1-deficient cells. NBS cells were labeled with IdU for 30 min, treated with 1 μm CPT or were mock-treated for 5 h, and frequency distributions of lengths of more than 100 DNA fibers from three independent experiments in each group were calculated. Inset shows western blot analysis for NBS1 expression in NBS and NBS+NBS1 cells. (B) Rate of nascent DNA strand degradation is slow in WS cells. WS cells were labeled with IdU for 30 min, treated with 1 μm CPT or were mock-treated for 1, 2.5, and 5 h, and frequency distributions of lengths of more than 100 DNA fibers from two-three independent experiments in each group were calculated. (C) Nascent DNA strands are degraded from 3′-5′ direction in the absence of WRN. HeLa cells transfected with WRN shRNA were first labeled with CldU then with IdU for 20 min each. Cells were then treated with 1 μm CPT or were mock-treated for 5 h, and the IdU:CldU ratios of more than 100 DNA fibers from two independent experiments in each group were calculated. (D) WRN blocks MRE11-dependent degradation of nascent DNA strands in response to replication-associated DSBs. WS cells were labeled with IdU for 30 min. Cells were then treated with or without 1 μM CPT and with or without 100 μM MRE11 exonuclease inhibitor mirin for 5 h. The frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. (E) WRN protects replication forks from MRE11-dependent degradation. WS cells were transfected with either control or MRE11 shRNA, labeled with IdU for 30 min, then treated with or without 1 μM CPT for 5 h. The frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. Inset shows western blot for MRE11 expression in WS cells transfected with control and MRE11 shRNAs. (F) WRN prevents MRE11-mediated degradation of replication forks. MRE11-defective ATLD cells were transfected with either control or WRN shRNA. At 72 h after the transfection, cells were labeled with IdU for 30 min, then treated with or without 1 μM CPT for 5 h. The frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. Inset shows western blots for WRN and MRE11 expression in ATLD cells.

Figure 6. WRN and Rad51 additively protect nascent DNA strands from MRE11-mediated degradation

(A) Nascent DNA strands are shortened in the absence of Rad51. HT1080 cells stably expressing tetracycline-inducible Rad51 shRNA were labeled with IdU for 30 min, then treated with or without 1 μM CPT for 5 h. The frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. Inset shows analysis of Rad51 expression in HT1080 cells with and without doxycycline treatment for 72 hours. (B) Rad51 protects replication forks from MRE11-mediated degradation in response to replication-associated DSBs. WS+WRN cells were pre-treated with 100 μM B02 for 8 h. Cells were labeled with IdU for 30 min and then treated with or without 1 μM CPT and with or without 100 μM mirin for 5 h. Subsequently, frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. (C) WRN and Rad51 additively protect nascent DNA strands from MRE11-mediated degradation. HT1080 cells stably expressing tetracycline-inducible Rad51 shRNA were transfected with WRN shRNA. At 72 h after the transfection, cells were labeled with IdU for 30 min and then treated with or without 1 μM CPT and with or without 100 μM mirin for 5 h. The frequency distributions of lengths of more than 100 DNA fibers from two-three independent experiments in each group were calculated. Inset shows western blot for Rad51 and WRN expression in HT1080 cells. (D) WRN and Rad51 additively block replication fork degradation mediated by MRE11. WS cells were pre-treated with B02 for 8 h. Cells were labeled with IdU for 30 min and then treated with or without 1 μM CPT and with or without 100 μM mirin for 5 h. The frequency distributions of lengths of more than 100 DNA fibers from two or three independent experiments in each group were calculated. See also Figure S5. (E) Stabilization of DNA-Rad51 complex in WS cells protects nascent DNA strands. WS cells were transfected with Rad51 K133R mutant, labeled with IdU for 30 min, then treated with or without 1 μM CPT for 5 h. The frequency distributions of lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. Inset shows over-expression of wild-type and K133R Rad51 in WS cells. See also Figure S5. (F) Over-expression of wild-type Rad51 in WS cells partially prevents nascent DNA strand degradation. WS cells were transfected with wild-type Rad51, labeled with IdU for 30 min, treated with or without 1 μM CPT for 5 h, and frequency distributions in lengths of more than 100 DNA fibers from two independent experiments in each group were calculated. Sell also Figure S5.

Figure 7. WRN, NBS1, Rad51 and MRE11 assemble onto nascent DNA strands to maintain genome stability in response to replication stress

(A) WRN, NBS1, Rad51 and MRE11 associate with nascent DNA strands. WS and WS+WRN cells were labeled with IdU for 60 min and then treated with 1 μM CPT for 5 hr. Cells were cross-linked with paraformaldehyde and the chromatin fraction (input) was subjected to co-immunoprecipitation using anti-BrdU mouse monoclonal antibodies. Western blots were probed with anti-WRN, anti-MRE11, anti-NBS1, anti-Rad51, and anti-Histone 3 antibodies. (B) A model depicting the choreography of WRN, NBS1, Rad51 and MRE11 action in the maintenance of nascent DNA strands in response to replication-associated DSBs. See also Figure S6.