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Int J Mol Sci
2024 Dec 05;2523:. doi: 10.3390/ijms252313097.
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Interaction of DDB1 with NBS1 in a DNA Damage Checkpoint Pathway.
Lim HE
,
Lim HJ
,
Yoo HY
.
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Various DNA damage checkpoint control mechanisms in eukaryotic cells help maintain genomic integrity. Among these, NBS1, a key component of the MRE11-RAD50-NBS1 (MRN) complex, is an essential protein involved in the DNA damage response (DDR). In this study, we discovered that DNA damage-binding protein 1 (DDB1) interacts with NBS1. DDB1 is a DDR sensor protein found in UV-induced DNA replication blocks. Through pull-down and immunoprecipitation assays conducted in Xenopus egg extracts and human cell lines, we demonstrated a specific interaction between NBS1 and DDB1. DDB1 was also found to associate with several proteins that interact with NBS1, including DNA topoisomerase 2-binding protein 1 (TopBP1) and Mediator of DNA damage checkpoint protein 1 (MDC1). Notably, the interaction between DDB1 and NBS1 is disrupted in MDC1-depleted egg extracts, indicating that MDC1 is necessary for this interaction. Furthermore, the depletion of DDB1 leads to increased Chk1 activation upon DNA damage. These novel findings regarding the interaction between NBS1 and DDB1 provide new insights into how DDB1 regulates DNA damage pathways.
Figure 1. The association of DDB1 with NBS1 in Xenopus egg extracts and human cells. (A) Interphase egg extracts containing NBS1-FLAG, with or without pA-pT, were subjected to immunoprecipitation using anti-FLAG antibodies. The resulting immunoprecipitates underwent SDS-PAGE followed by silver staining. The boxed region of the gel was further analyzed by mass spectrometry. Lane 1 displays molecular mass markers. (B) Anti-FLAG antibody beads lacking recombinant proteins (lanes 1 and 2) and those containing NBS1-FLAG (lanes 3 and 4) were incubated in egg extracts with or without pA-pT. The beads were collected and analyzed via immunoblotting using anti-DDB1 and anti-NBS1 antibodies. (C) Anti-FLAG antibody beads devoid of recombinant proteins (lanes 1 and 2) and those with DDB1-FLAG (lanes 3 and 4) were incubated in egg extracts with or without pA-pT. After isolation, the beads were immunoblotted with anti-NBS1 and anti-DDB1 antibodies. (D) Control immunoprecipitates and anti-NBS1 IPs from egg extracts, with or without pA-pT, were analyzed by immunoblotting for DDB1 and TopBP1. (E) HEK 293T cells were transfected with either a control plasmid or vectors that express HA-tagged human DDB1 (HA-DDB1) and FLAG-tagged human NBS1 (NBS1-FLAG). Following a 48 h incubation, the cells received either mock treatment or were exposed to IR (10 Gy). After 2 h, cell lysates were collected and anti-FLAG immunoprecipitations were performed and subsequently analyzed through immunoblotting using anti-HA and anti-FLAG antibodies, while the cell lysates were examined with the specified antibodies. (F) HEK 293T cells were transfected with either a control vector or vectors expressing FLAG-tagged human NBS1 (NBS1-FLAG) and HA-tagged human DDB1 (HA-DDB1). Cell lysates were prepared as described in (E). Anti-HA IP from these lysates was immunoblotted for the specified proteins.
Figure 2. DDB1 interaction with TopBP1 in Xenopus egg extracts and human cells. (A) Anti-FLAG antibody beads lacking recombinant proteins (lanes 1 and 2) and those containing TopBP1-FLAG (lanes 3 and 4) were incubated with egg extracts, both with and without pA-pT. The beads were collected and subjected to immunoblotting using anti-DDB1 and anti-NBS1 antibodies. (B) HEK 293T cells were either mock-treated or subjected to IR (10 Gy). At 2 h after treatment, cell lysates were obtained. Immunoprecipitation with control IgG and anti-DDB1 from these lysates was followed by immunoblotting with anti-DDB1, anti-TopBP1, and anti-NBS1 antibodies. The corresponding antibodies were used to analyze the cell lysates as well. (C) HEK 293T cells were transfected with either a control plasmid or vectors that express HA-tagged human DDB1 (HA-DDB1) and FLAG-tagged human TopBP1 (FLAG-TopBP1). Cell lysates were prepared as outlined in (B). Immunoprecipitation with anti-FLAG antibodies from the cell lysates was subsequently analyzed by immunoblotting for the specified proteins.
Figure 3. MDC1 mediates the interaction between NBS1 and DDB1. (A) Immunoprecipitates (IP) were obtained using control antibodies (lanes 1 and 2) and anti-MDC1 antibodies (lanes 3 and 4) from egg extracts, with or without pA-pT, and were subsequently analyzed by immunoblotting using anti-DDB1, anti-NBS1, and anti-TopBP1 antibodies. (B) Egg extracts containing sperm nuclei were incubated for 60 min either without EcoRI (lane 2) or in the presence of EcoRI (lanes 1 and 3). After this incubation, nuclear fractions were prepared and treated with protein A magnetic beads containing either control antibodies (lane 1) or anti-MDC1 antibodies (lanes 2 and 3). The beads were collected and analyzed via immunoblotting for DDB1, NBS1, and TopBP1. (C) HEK 293T cells were transfected with a control plasmid or a plasmid that expresses HA-tagged human MDC1 (HA-MDC1). After 48 h, the cells received either mock treatment or were subjected to IR at 10 Gy. Two hours later, cell extracts were prepared, followed by anti-HA immunoprecipitation and immunoblotting with antibodies against DDB1, TopBP1, NBS1, and MDC1. The cell lysates were also analyzed with specified antibodies. (D) Egg extracts were either mock-depleted using control antibodies (lane 1) or subjected to immunodepletion with anti-MDC1 antibodies (lane 2). Following this, the extracts were processed for immunoblotting using antibodies against MDC1 and NBS1. (E) Egg extracts that were either mock-depleted (lanes 1 and 2) or depleted of MDC1 (lanes 3 and 4), with or without the presence of pA-pT, were incubated with anti-FLAG antibody beads containing DDB1-FLAG. The beads were then collected again and subjected to immunoblotting using anti-NBS1 and anti-DDB1 antibodies.
Figure 4. Characterization of DDB1 binding sites on NBS1 and MDC1. (A) Anti-FLAG antibody beads that included no recombinant protein (lanes 1 and 2), NBS1-FLAG(1–410) (lanes 3 and 4), or NBS1-FLAG(338–762) (lanes 5 and 6) were incubated in egg extracts with or without of pA-pT. After isolating the beads, they were analyzed by immunoblotting with anti-DDB1 and anti-FLAG antibodies. (B) Anti-FLAG antibody beads that were either free of recombinant protein or contained DDB1-FLAG(1–600) or DDB1-FLAG(590–1140) were incubated in egg extracts, both in the presence and absence of pA-pT. After incubation, the beads were collected again and analyzed by immunoblotting with anti-NBS1 and anti-FLAG antibodies. (C) Anti-FLAG antibody beads that lacked recombinant proteins (lanes 1 and 2) or contained MDC1-FLAG(1–1166) (lanes 3 and 4) and MDC1-FLAG(989–2100) (lanes 5 and 6) were incubated in egg extracts with or without pA-pT. The beads were collected and immunoblotted using anti-DDB1 and anti-FLAG antibodies. (D) Recombinant DDB1-FLAG was immobilized on anti-FLAG antibody beads (lanes 5 and 6) and incubated with egg extracts containing GST-MDC1(1–350) in both the presence and absence of pA-pT. The beads were re-isolated and analyzed for GST by immunoblotting. (E) Recombinant DDB1-FLAG on anti-FLAG antibody beads were incubated with egg extracts containing GST-MDC1(1–130), GST-MDC1(110–260), or GST-MDC1(250–350) with or without pA-pT. After retrieval, the beads were immunoblotted with anti-GST antibodies.
Figure 5. The role of DDB1 in regulating Chk1 activation in response to DNA damage. (A) Egg extracts containing sperm nuclei were treated under three conditions: without any additives (lanes 1 and 4), with 0.05 U/μL EcoRI (lanes 2 and 5), or with 50 μg/mL aphidicolin (Aph) (lanes 3 and 6). The nuclear fractions (lanes 1–3) and chromatin fractions (lanes 4–6) were analyzed by SDS-PAGE followed by immunoblotting for MDC1, TopBP1, NBS1, DDB1, and Orc2. EcoRI induces double-stranded DNA breaks, while aphidicolin causes DNA replication blockage. (B) Egg extracts were treated with control antibodies for mock depletion (lanes 1 and 2) or subjected to immunodepletion using anti-DDB1 antibodies (lanes 3 and 4). Both mock-depleted and DDB1-depleted extracts containing sperm nuclei were incubated for 90 min, either without treatment or with EcoRI or aphidicolin (APH). Subsequently, nuclear fractions were isolated and immunoblotted with antibodies specific for Xenopus DDB1, phospho-Ser-344 of Chk1, Xenopus Chk1, and Xenopus Chk2. (C) HeLa cells were transfected with either control siRNA or DDB1 siRNA1. They were then either mock-treated (lanes 1 and 3) or exposed to IR (10 Gy) (lanes 2 and 4). Two hours post-treatment, cell lysates were prepared and analyzed via immunoblotting with the specified antibodies. The phospho-Chk1 signals (lane 4) were quantified and normalized against those in lane 2, with results displayed beneath each band. (D) The graph illustrates the densitometric analysis results from the immunoblots, showing normalized phospho-Chk1 levels in DDB1 siRNA-treated cells compared to control siRNA-treated cells. The data come from four independent experiments and are presented as mean ± SD. (E) Proposed model for the interactions among MDC1, TopBP1, DDB1, and the MRN complex. MDC1 facilitates the connection between DDB1 and NBS1 within the MRN complex.
