Structural characterization of human RPA70N association with DNA damage response proteins
- School of Public Health & Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, China
Figures
Figure 1 with 1 supplement
RPA70N–peptide complex structures determined in this study.
(A).Linear domain diagram of the RPA heterotrimer. (B) Ribbon representation of human RPA70N from PDB 5EAY. L12 denotes the loop between β1 and β2, L45 is the loop between β4 and β5. (C) Surface …
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Figure 1—source data 1
Data collection and refinement statistics.
- https://cdn.elifesciences.org/articles/81639/elife-81639-fig1-data1-v1.docx
Figure 1—figure supplement 1
Sequence alignment and omit maps of bound peptides (related to Figure 1).
(A) Alignment of human RPA70N-interacting motif sequences from different proteins. (B) Linker and peptide sequences used in RPA70N fusion constructs. (C–K) mFo-DFc electron density maps (green mesh) …
Figure 2 with 1 supplement
Structure of the RPA70N–HelB complex.
(A) Linear domain diagram of HelB showing the position and sequence of the RPA70N interacting motif. SLD: subcellular domain. (B) Structure of RPA70N in complex with HelB, showing the surface …
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Figure 2—source data 1
Raw data of all western blots from Figure 2.
- https://cdn.elifesciences.org/articles/81639/elife-81639-fig2-data1-v1.zip
Figure 2—figure supplement 1
Characterization of RPA70N–HelB interaction (related to Figure 2).
(A) Crystal packing analysis indicates that a HelB peptide from one RPA70N–HelB fusion molecule binds to the RPA70N portion of a neighboring molecule in the crystal. (B–D) ITC titration profiles of …
Figure 3 with 2 supplements
Structures of two RPA70N–BLM complexes.
(A) Linear domain diagram of BLM showing the position and sequence of RPA70N interacting motifs. DHBN, dimerization helical bundle in N-terminal region; RDC, RecQ-conserved domain; HRDC, helicase …
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Figure 3—source data 1
Raw data from all western blots shown in Figure 3.
- https://cdn.elifesciences.org/articles/81639/elife-81639-fig3-data1-v1.zip
Figure 3—figure supplement 1
Characterization of RPA70N–BLM interaction (related to Figure 3).
(A) Surface representation of the two RPA70N molecules interacting with a BLMp2 peptide, which is displayed as magenta sticks. (B–D) ITC titration profiles of (B) WT BLMp2 (550–570 aa) peptide, (C) …
Figure 3—figure supplement 2
Characterization of the RPA70N–BLM interaction (related to Figure 3).
(A) HeLa cells expressing EGFP–BLM, EGFP–BLM (W154A/F160A, p1M), EGFP–BLM (D560A/F561A/D562A, p2M) or EGFP–BLM (W154A/F160A/D560A/F561A/D562A, p1p2M) were treated with HU (2 mM, 3 h), fixed and …
Figure 4 with 1 supplement
Structure of the RPA70N–RMI1 complex.
(A) Linear domain diagram of RMI1, showing the position and sequence of the RPA70N interacting motif. (B) Ribbon representation of the RPA70N–RMI1 crystal structure, the RMI1 peptide is coordinated …
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Figure 4—source data 1
Raw data for all all western blots shown in Figure 4.
- https://cdn.elifesciences.org/articles/81639/elife-81639-fig4-data1-v1.zip
Figure 4—figure supplement 1
Characterization of the RPA70N–RMI1 interaction (related to Figure 4).
(A) Surface representation of the two RPA70N molecules interacting with an RMI1 peptide, which is displayed as olive-colored sticks. (B–C) ITC titration profiles of WT RMI1 (243–262aa) peptide (B) …
Figure 5 with 1 supplement
Structure of the RPA70N–WRN complex.
(A) Linear domain diagram of WRN showing the position and sequence of RPA70N-interacting motifs. (B) Ribbon representation of the RPA70N–WRN crystal structure. The fused WRN peptide forms an α helix …
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Figure 5—source data 1
Raw data for all of the western blots shown in Figure 5.
- https://cdn.elifesciences.org/articles/81639/elife-81639-fig5-data1-v1.zip
Figure 5—figure supplement 1
Characterization of the RPA70N–WRN interaction (related to Figure 5).
(A) Ribbon representation of the two WRN peptides interacting with RPA70N. WRNa (purple-blue) binds to the groove, while WRNb (light green) binds to the side pocket with Y436b.(B) Ribbon …
Figure 6 with 1 supplement
Structure of the RPA70N–ATRIP complex.
(A) Linear domain diagram of ATRIP, showing the position and sequence of the RPA70N-interacting motif. CC, coiled-coiled domain; ATR-BD, ATR binding domain. (B) Ribbon representation of the …
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Figure 6—source data 1
Raw data for all of the western blots shown in Figure 6.
- https://cdn.elifesciences.org/articles/81639/elife-81639-fig6-data1-v1.zip
Figure 6—figure supplement 1
Characterization of the RPA70N–ATRIP interaction (related to Figure 6).
(A–C) ITC titration profiles of WT ATRIP peptide (53–69aa) (A), F55A mutant peptide (B) or F55A/L60A/L63A mutant peptide (C) with RPA70N. (D) ITC titration data for WT RPA70N (1–120aa), R91A or …
Figure 7 with 1 supplement
Structure of the RPA70N–MRE11 complex.
(A) Linear domain diagram of MRE11, showing the position and sequence of the RPA70N-interacting motif. (B) Ribbon representation of the RPA70N–MRE11 crystal structure. The MRE11 peptide is colored …
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Figure 7—source data 1
Raw data for all of the western blots shown in Figure 7.
- https://cdn.elifesciences.org/articles/81639/elife-81639-fig7-data1-v1.zip
Figure 7—figure supplement 1
Characterization of the RPA70N–MRE11 interaction (related to Figure 7).
(A) ITC titration data for WT RPA70N (1–120aa) and R91A or R41A/R43A/R91A mutant proteins with MRE11 peptide (538–563aa). (B–C) ITC titration profiles of RPA70N R91A mutant protein (B) and …
Figure 8 with 1 supplement
Structure of the RPA70N–RAD9 complex.
(A) Linear domain diagram of RAD9, showing the position and sequence of the RPA70N-interacting motif. IDCL, interdomain connecting loop. (B) Ribbon representation of the RPA70N–RAD9 crystal …
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Figure 8—source data 1
Raw data for all of the western blots shown in Figure 8.
- https://cdn.elifesciences.org/articles/81639/elife-81639-fig8-data1-v1.zip
Figure 8—figure supplement 1
Characterization of the RPA70N–RAD9 interaction (related to Figure 8).
(A–B) ITC titration profiles of WT RAD9 (296–314aa) peptide (A) and F298A/M307A/I303A mutant peptide (B) with RPA70N. (C) ITC titration data for WT RPA70N (1–120aa) and for R31H, R91A or …
Figure 9 with 1 supplement
Structure of the RPA70N–ETAA1 complex.
(A) Linear domain diagram of ETAA1, showing the position and sequence of the RPA70N-interacting motif. ETAA1 has a coiled-coiled domain (CC) and two RPA-binding motifs (RBM1, RBM2). (B) Ribbon …
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Figure 9—source data 1
Raw data for all of the western blots shown in Figure 9.
- https://cdn.elifesciences.org/articles/81639/elife-81639-fig9-data1-v1.zip
Figure 9—figure supplement 1
Characterization of the RPA70N–ETAA1 interaction (related to Figure 9).
(A–B) ITC titration profiles of the WT ETAA1 (599–622 aa) peptide (A) and the W600A/L610A/Y611A mutant peptide (B) with RPA70N. (C) ITC titration data for WT RPA70N (1-120aa) and for R31H, R91A or …
Figure 10 with 1 supplement
Comparison of RPA70N–peptide complex structures.
(A) Summary of the RPA70N-binding proteins for which RPA70N complex structures are available (Ddc2 binds to Rfa1N). (B) Superposition of apo RPA70N (orange, PDB: 2B29) with RPA70N structures …
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Figure 10—source data 1
Editable word file of Figure 10A.
- https://cdn.elifesciences.org/articles/81639/elife-81639-fig10-data1-v1.docx
Figure 10—figure supplement 1
Model of protein recruitment in the RPA70N-mediated DNA damage response (related to Figure 10).
(A) A simplified model showing how exposed ssDNA attracts RPA molecules, which recruit many DNA damage response proteins including ATR/ATRIP, the 9-1-1 clamp, MRN, ETAA1 and TOPBP1 to the stalled …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | Rosetta 2(DE3) chemically competent cell | Novagen | 71402 | |
| Strain, strain background (E. coli) | E. coli BL21(DE3) cells | Novagen | 69450 | |
| Strain, strain background (E. coli) | Trelief 5α chemically competent cell | TSINGKE | TSC-C01-100 | |
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216 | Authenticated by STR profiling, no mycoplasma contamination |
| Cell line (H. sapiens) | HeLa | Cell bank of the Chinese Academy of Sciences | SCSP-504 | Authenticated by STR profiling, no mycoplasma contamination |
| Antibody | Anti-GFP rabbit monoclonal antibody | Beyotime | AF1483 | WB (1:5000) |
| Antibody | Anti-RPA32 rabbit monoclonal antibody | Beyotime | AG3115 | WB (1:2000), IF (1:50) |
| Antibody | Anti-β-actin mouse monoclonal antibody | Proteintech | 66009–1-Ig | WB (1:5000) |
| Antibody | Anti-flag tag mouse monoclonal antibody | Beyotime | AF2852 | IF (1:50) |
| Antibody | Horseradish peroxidase labelled goat anti-mouse polyclonal secondary antibody | Beyotime | A0216 | WB (1:5000) |
| Antibody | Horseradish peroxidase labelled goat anti-rabbit polyclonal secondary antibody | Beyotime | A0208 | WB (1:5000) |
| Antibody | Alexa 568 goat anti-rabbit polyclonal antibody IgG (H+L) cross-adsorbed secondary antibody | Thermo Fisher Scientific | A-11036 | IF (1:400) |
| Antibody | Alexa 488 goat anti-mouse polyclonal antibody IgG (H+L) cross-adsorbed secondary antibody | Thermo Fisher Scientific | A32723 | IF (1:400) |
| Recombinant DNA reagent | pcDNA3.1(+) | Invitrogen | V79020 | |
| Recombinant DNA reagent | pRSFDuet-1 vector | Novagen | 71341 | |
| Commercial assay or kit | ClonExpress II One Step Cloning Kit | Vazyme | C112-01/02 | |
| Chemical compound, drug | Hydroxyurea | Beyotime | Cat#S1961 | |
| Chemical compound, drug | (S)-(+)-Camptothecin, 98% | J&K | Cat#7689-03-4 | |
| Chemical compound, drug | 2-(4-Amidinophenyl)–6-indolecarbamidine dihydrochloride | Beyotime | C1006 | DAPI staining solution used to stain nucleus |
