BID interacts with the N-terminal domain of RPA70 in vitro. (A) Schematic diagram of the functional domains of RPA. The interactions between BID and various RPA domains are summarized. (B) BID interacts with RPA. Purified recombinant mouse BID and His-tagged RPA were incubated in binding buffer at room temperature for 30 min. BID was immunoprecipitated using anti-BID antibody, and the immunoprecipitated proteins were resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies. (C) BID interacts with RPA70. Purified recombinant mouse BID was incubated with His-tagged RPA70-PDI fusion protein, His-tagged RAP70NAB domain, or His-tagged RPA70AB domain as in panel B. BID was immunoprecipitated using anti-BID antibody, and the immunoprecipitated proteins were resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies. (D) BID interacts with the N domain of RPA70. Purified recombinant mouse BID and His-tagged RPA70N domain (amino acids [aa] 1 to 168) were incubated as in panel B. BID was immunoprecipitated using anti-BID antibody, and the immunoprecipitated proteins were resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies. (E) BID does not interact with the C domain of RPA32. Purified recombinant mouse BID and His-tagged RPA32C domain were incubated as in panel B. BID was immunoprecipitated using anti-BID antibody, and the immunoprecipitated proteins were resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies. (F) BID does not interact with RPA32/RPA14 domains. Purified recombinant mouse BID and RPA32/RPA14 subunits were incubated as in panel B. BID was immunoprecipitated using anti-BID antibody, and the immunoprecipitated proteins were resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies.

NMR analysis and model of the interaction of RPA70N and BID. (A) Identification of the RPA70N residues in the BID binding site. Overlaid ¹⁵N-¹H HSQC spectra of ¹⁵N-labled RPA70N (aa 1 to 120) in the absence (black) and presence (red) of BID. (B) Map of RPA70N residues (yellow) perturbed upon the addition of BID on the structure of RPA70N. (C) Identification of the BID residues in the RPA70N interaction domain. Binding of RPA70N with BID monitored on the ¹⁵N-¹H HSQC spectra of ¹⁵N-labled BID in the absence (black) and presence (red) of RPA70N. (D) Map of BID residues (yellow) perturbed upon the addition of RPA70N on the structure of BID. (F) Electrostatic complementarity of the RPA70N (top) and BID (bottom) binding sites. (E) NMR-based model of the complex of BID with RPA70N. Ribbon representations of PA70N and BID are colored in salmon red and cyan, respectively. (G) A close-up view of the model of the BID-RPA70N complex.

Mutations in the RPA-ID of BID or the basic cleft region of RPA70N impair BID-RPA70N interaction. (A) BID interacts with the basic cleft of RPA70N. Purified recombinant His-tagged wild-type RPA70N or RPA70N mutated at residues 41, 43, 91, and 92, charged residues located around the RPA basic cleft region, was incubated with BID in binding buffer at room temperature for 30 min. BID was immunoprecipitated using anti-BID antibody, and the immunoprecipitated proteins were resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies. (B) Schematic diagram of the RPA-ID and IH5-mutated BID. Glu121, Asp122, and Asp126 in the RPA-ID region of BID are marked. (C) The BID RPA-ID region interacts with RPA70N. Purified recombinant His-tagged wild-type RPA70N was incubated with wild-type, RPA-ID-, or IH5-mutated mouse BID in binding buffer at room temperature for 30 min. BID was immunoprecipitated using anti-BID antibody, and the immunoprecipitated proteins were resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies. (D) Purified recombinant His-tagged wild-type RPA70N was incubated with wild-type, RPA-ID-, or IH5-mutated human BID in binding buffer at room temperature for 30 min. BID was immunoprecipitated using anti-BID antibody, and the immunoprecipitated proteins were resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies. (E) Mutation in the RPA-ID region of BID impairs BID-RPA interaction in U2OS cells. U2OS cells harboring stably expressed HA-tagged wild-type hBID or mutated hBID in RPA-ID or the IH5 region were treated with 10 mM hydroxyurea for 5 h. Then, the expressed HA-tagged BID was immunoprecipitated from purified nuclear extracts by anti-HA antibody and RPA70 was detected in the immunoprecipitated products. A solid arrow denotes HA-tagged BID. A dashed arrow denotes endogenous BID. (F) Biotinylated ssDNA was incubated with streptavidin beads for 30 min at 4°C. Then, the pulldown products were incubated with His-tagged RPA in the presence of wild-type or mutated mouse BID for 1 h at 4°C and the pulldown products were resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies.

RPA-ID of BID is important for normal ATR function following replicative stress. (A) An intact RPA-ID region of BID is required for normal recovery of DNA synthesis following hydroxyurea withdrawal. U2OS cells overexpressing HA-tagged wild-type, RPA-ID-, or IH5-mutated human BID were transfected with BID siRNA for 72 h. Then, cells were treated with 10 mM hydroxyurea overnight and released into fresh medium containing 1 μg/ml nocodazole for the indicated times. Cells were fixed and stained with propidium iodide. Live cells were analyzed by flow cytometry gated on forward- and side-scatter channels (FSC/SSC). The quantitative analysis of the arrested G₁-/early-S-phase cells following a 32-h HU withdrawal is shown in panel B. *, P < 0.05. (C) An intact RPA-ID region of BID is important for normal ATR substrate phosphorylation following hydroxyurea treatment. U2OS cells overexpressing HA-tagged wild-type BID or RPA-ID-mutated human BID were transfected with BID siRNA for 72 h. Silent mutations were introduced into the BID siRNA-targeted region so that only endogenous BID was knocked down by BID siRNA. Then, cells were treated with 10 mM hydroxyurea for 5 h, and protein extracts were resolved on SDS-PAGE gels and immunoblotted with anti-phospho-CHK1 and anti-RPA32. (D) U2OS cells overexpressing HA-tagged wild-type, RPA-ID, or IH5 RPA-ID-mutated human BID were transfected with BID siRNA for 72 h. Silent mutations were introduced into the BID siRNA-targeted region so that only endogenous BID was knocked down by BID siRNA. Then, cells were treated with 10 mM hydroxyurea overnight. The untreated and treated cells were collected in ice-cold PBS and detected in an alkaline comet assay (Trevigen). The samples were run in alkaline electrophoresis solution at 21 V for 30 min. At least 60 randomly chosen comets per sample were analyzed by CometScore program version 1.5. *, P < 0.05.

Mutation in the RPA-ID region of BID does not significantly alter BID's apoptotic function in the extrinsic cell death pathway. (A) Caspase 8 cleaves RPA-ID-mutated BID. Purified wild-type and RPA-ID-mutated mouse BID were incubated with active caspase 8 (Millipore) in vitro for 0.5, 1, and 2 h. The reaction products were resolved by SDS-PAGE and immunoblotted with anti-BID antibody. tBID, caspase-cleaved BID. (B) Incubation with RPA does not affect BID's sensitivity to caspase 8 in vitro. Purified wild-type mouse BID was incubated with active caspase 8 in the presence of RPA or BSA for 20, 40, and 60 min. The reaction products were resolved by SDS-PAGE and immunoblotted with anti-BID antibody. (C) Caspase-cleaved BID (tBID) binds with RPA in vitro. Purified recombinant mouse BID was first incubated with activated caspase 8 for 4 h at 37°C. Then, tBID was incubated with purified recombinant His-tagged RPA70-PDI fusion protein, and in vitro co-IP assay was performed. (D) RPA-ID-mutated BID binds to other BCL-2 family proteins. 293T cells were transfected with wild-type BID or RPA-ID-mutated mouse BID for 48 h. Then, BID was immunoprecipitated from whole-cell extracts with anti-BID antibody. The immunoprecipitated products were resolved by SDS-PAGE followed by immunoblotting with anti-BID, anti-MCL1, and anti-BCL2 antibodies. (E) U2OS cells overexpressing HA-tagged wild-type or RPA-ID-mutated human BID were lysed, and HA-tagged BID was immunoprecipitated from whole-cell extracts with anti-HA antibody. The immunoprecipitated products were resolved by SDS-PAGE followed by immunoblotting with anti-BID, anti-MCL1, and anti-BCL2 antibodies. A solid arrow denotes HA-BID, and a dashed arrow denotes endogenous BID. (F) Cells harboring RPA-ID-mutated BID show similar sensitivities to TRAIL/CHX treatment. U2OS cells overexpressing HA-tagged wild-type or RPA-ID-mutated human BID were transfected with BID siRNA for 72 h. Then, cells were treated with 50 ng/ml TRAIL and 5 μg/ml CHX for the indicated times. The apoptotic cells were detected using the annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BioVision) according to the manufacturer's instructions.

Stability of the replication fork is diminished in BID-deficient cells following replicative stress. (A) U2OS cells were transfected with control siRNA or BID siRNA for 72 h. Then, cells were treated with 10 mM hydroxyurea over time, and the chromatin fraction was purified and resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies to various factors involved in the replication fork. (B) U2OS cells overexpressing HA-tagged wild-type hBID, RPA-ID-mutated hBID, or helix 4-mutated hBID were transfected with BID siRNA for 72 h. Overexpressed BID and BID mutants harbor silent mutations in the BID siRNA-targeted region to evade siRNA knockdown, and thus only endogenous BID was knocked down by BID siRNA. Then, cells were treated with 10 mM hydroxyurea over time, and the chromatin fraction was purified, resolved on SDS-PAGE gels, and immunoblotted with the indicated antibodies. (C) U2OS cells used in B, overexpressing HA-tagged wild-type hBID, RPA-ID-mutated hBID, or helix 4-mutated hBID harboring silent mutations to evade siRNA knockdown, were transfected with BID siRNA for 72 h. Then, cells were treated with 10 mM hydroxyurea for 5 h, and the chromatin fraction was purified, resolved on SDS-PAGE gels, and immunoblotted with anti-BID antibody.

BID facilitates the RPA-ATRIP interaction in vitro in a dose-dependent manner. (A) His-GST fused wild-type RPA70N or RPA70N harboring R41E/R43E mutations (250 pmol) was bound to glutathione-agarose beads. Then, the nuclear fraction was purified from U2OS cells stably expressing HA-tagged ATRIP. The nuclear lysate was incubated with the RPA70N or RPA70N-R41E/R43E beads in the presence of 50 or 1,250 pmol of purified human BID at room temperature for 1 h. The GST pulldown product was resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies. (B) His-GST fused wild-type RPA70N (250 pmol) was bound to glutathione-agarose beads. Then, U2OS cells stably expressing HA-tagged ATRIP were left untreated or were treated with 10 mM HU for 5 h, and the nuclear fraction was purified. The nuclear lysate was incubated with the RPA70N glutathione-agarose beads in the presence of 10, 50, 250, and 1,250 pmol of purified human BID at room temperature for 1 h. The GST pulldown product was resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies. (C) His-GST fused wild-type RPA70N (250 pmol) was bound to glutathione-agarose beads. Then, the nuclear fraction was purified from U2OS cells stably expressing HA-tagged ATRIP. The nuclear lysate was incubated with the RPA70N glutathione-agarose beads in the presence of 10 pmol of purified wild-type mouse BID or BID mutated in the RPA-ID, IH5, H4A (L105C/L109C), and H4B (Q112A/N115A) regions at room temperature for 1 h. The GST pulldown product was resolved on SDS-PAGE gels and immunoblotted with the indicated antibodies. (D) A model of representing the regions of BID that interact with proteins in the DNA damage sensor complex to mediate DNA damage response and the region of BID that interacts with proteins in the BCL-2 family to mediate apoptosis.