dk02  (Dojindo Labs)

Journal: Molecular cell

Article Title: RAD51 protects abasic sites to prevent replication fork breakage.

doi: 10.1016/j.molcel.2024.07.004

Figure Lengend Snippet: Figure 1. RAD51 binds to ssDNA containing abasic sites (A) Surface plasmon resonance analysis of RAD51’s interaction with abasic ssDNA. The bio- tinylated DNA ligand was coupled to a streptavi- din-coated sensor chip, and the RAD51 analyte was added to the mobile phase to measure the RU response. The left panel shows representative sensorgrams of RAD51 binding to DNA oligonu- cleotides with 1, 3, and 5 AP sites, and no AP sites as control. The right panel reports the apparent association rates (Kon) of RAD51 to the abasic DNA oligonucleotides, from sensogram data analysis. (B) Cryo-EM structure of a RAD51 nucleoprotein filament with ssDNA containing multiple, periodi- cally spaced AP sites (AP7). The RAD51 protomers in the filament are drawn as ribbons and the ssDNA is in spacefill representation. (C) Cryo-EM density of the ssDNA in the filament, with fitted oligonucleotide structure. The abasic nucleotides are colored pink and their position is indicated by an arrow. The DNA sequence is shown next to the structure; abasic site positions are marked by x. (D) RAD51’s DNA-binding loops L1 and L2 inter- action with the AP site. Loops are drawn as light blue ribbons, while the DNA is in light-brown filled- ring representation with the abasic nucleotide colored pink. (E) Superposition of pre-synaptic RAD51 NPF (PDB: 8BQ2) with the NPF on abasic ssDNA (this work). Two rotated views of the filament are shown, centered on the position of the AP site. See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER xAPE1 protein This manuscript N/A GST-BRC4 Hashimoto et al.14 N/A Geminin Aze et al.85 N/A POLq protein full length Mann et al.15 N/A Critical commercial assays CellTiter-Glo Luminescent Cell Viability Assay Promega Cat#G7571 Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye, Alexa Fluor 647 dye ThermoFisher Cat#C10337; CHEBI:52661 Cat#C10340; CRISPR Gene Knockout Kit v2 targeting APEX1 Sinthego N/A DNeasy Blood & Tissue Kit Qiagen Cat#69504; Lipofectamine CRISPRMAX Transfection Reagent ThermoFisher Cat#CMAX00003; Nucleostain- DNA Damage Quantification Kit -AP Site Counting Dojindo Cat#DK02-12; Qubit dsDNA HS Assay Kits ThermoFisher Scientific Cat#Q32854; RNAiMax ThermoFisher Cat#13778150; Deposited data Atomic Coordinates and CryoEM maps Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) PDB: 8RCD, 8RCF;EMDB: EMD-19050, EMD-19051 Images and graphs Mendeley Mendeley Data: https://doi.org/10.17632/2gjvrzddv7.1 Experimental models: Cell lines Human DLD-1 wild-type Horizon Cat#HD PAR-008; RRID:CVCL_0248 Human DLD-1 BRCA2-/- Horizon Cat#HD 105-007; RRID:CVCL_HD57 Human DLD-1 APEX1-/- This manuscript N/A Human DLD-1 DNMT1NA Scelfo et al.45 N/A Human DLD-1 DNMT3B-/- DNMT1NA Scelfo et al.45 N/A Human PEO1 BRCA2Y1655X Panzarino et al.41 N/A Human C4-2 PEO1 BRCA2WT Panzarino et al.41 N/A Experimental models: Organisms/strains Xenopus laevis females Nasco LM00535MX Xenopus laevis males Nasco LM00715MX Oligonucleotides siAPOBEC3B Horizon Cat#L-017432-00-0005 siBRCA2 Horizon Cat#L-003462-00-0010 siHMCES Sigma Cat#EHU036741-5UG siMRE11A Horizon Cat#L-009271-00-0010 siPRIMPOL Horizon Cat#L-016804-02-0005 siSMUG1 Horizon Cat#L-012838-01-0010 siTDG Horizon Cat#L-003780-01-0010 siTET2 Horizon Cat#L-013776-03-0010 Universal control siRNA Sigma-Aldrich Cat#SIC001 siRAD51 Horizon Cat#L-003530-00-0010 Recombinant DNA 6His-pET-SMUG1 This manuscript N/A pGEX-6P-1-APE1 This manuscript N/A Mre11 BACMID vector pTP2620 Bhaskara et al.33 Addgene Cat#113311; RRID:Addgene_113311 Rad50 BACMID vector pTP813 Bhaskara et al.33 Addgene Cat#113308; RRID:Addgene_113308 RAD51 Appleby et al.23 N/A (Continued on next page) Molecular Cell 84, 3026–3043.e1–e11, August 22, 2024 e2

Techniques: SPR Assay, Binding Assay, Control, Cryo-EM Sample Prep, Sequencing

Journal: Molecular cell

Article Title: RAD51 protects abasic sites to prevent replication fork breakage.

doi: 10.1016/j.molcel.2024.07.004

Figure Lengend Snippet: Figure 4. RAD51-mediated protection ag- ainst MRE11-dependent abasic DNA pro- cessing and fork breakage (A) Representative EM image of a replication fork from egg extract treated with APE1i (10 mM, 45 min). Magnified area in rectangle. Cartoon dotted lines show ssDNA. (B) Quantification of total ssDNA length for each fork isolated from extracts treated as indicated. Horizontal bars indicate the mean ± SD for a total of 72 forks. Overlay dots show n = 3 inde- pendent biological replicate means. One-way ANOVA, followed by Tukey’s post hoc test for multiple comparisons; **p < 0.01; ***p < 0.001; ****p < 0.0001. (C and D) Representative EM images of forks from an extract treated with APE1i (10 mM) and GST- BRC4 (0.5 mg/mL, 45 min). (E) Percentage of broken forks from extracts treated as indicated. Horizontal bars indicate mean ± SD relative to 72 forks. Overlay dots show n = 3 independent biological replicate means. One- way ANOVA, followed by Tukey’s post hoc test; **p < 0.01; ***p < 0.001; ****p < 0.0001. (F) EM of a rescued fork from extract treated with APE1i, GST-BRC4, and PFM01 (100 mM). Base pair lengths numbered; gaps by red arrows; broken arm by black arrow. (G) Denaturing gel showing fluorescein-labeled ssDNA with an AP-site generated by UDG-medi- ated uracil removal and treatment with buffer or 200 nM MR complex and increasing RAD51. (H) AP oligo cleavage percentage in the presence of MR and increasing RAD51. No RAD51 sample was taken as reference. Horizontal bars indicate mean ± SD of all data points. Overlay dots show n = 3 independent replicates. One-way ANOVA, followed by Dunnett’s post hoc test; ****p < 0.0001; ns, non-significant. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER xAPE1 protein This manuscript N/A GST-BRC4 Hashimoto et al.14 N/A Geminin Aze et al.85 N/A POLq protein full length Mann et al.15 N/A Critical commercial assays CellTiter-Glo Luminescent Cell Viability Assay Promega Cat#G7571 Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye, Alexa Fluor 647 dye ThermoFisher Cat#C10337; CHEBI:52661 Cat#C10340; CRISPR Gene Knockout Kit v2 targeting APEX1 Sinthego N/A DNeasy Blood & Tissue Kit Qiagen Cat#69504; Lipofectamine CRISPRMAX Transfection Reagent ThermoFisher Cat#CMAX00003; Nucleostain- DNA Damage Quantification Kit -AP Site Counting Dojindo Cat#DK02-12; Qubit dsDNA HS Assay Kits ThermoFisher Scientific Cat#Q32854; RNAiMax ThermoFisher Cat#13778150; Deposited data Atomic Coordinates and CryoEM maps Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) PDB: 8RCD, 8RCF;EMDB: EMD-19050, EMD-19051 Images and graphs Mendeley Mendeley Data: https://doi.org/10.17632/2gjvrzddv7.1 Experimental models: Cell lines Human DLD-1 wild-type Horizon Cat#HD PAR-008; RRID:CVCL_0248 Human DLD-1 BRCA2-/- Horizon Cat#HD 105-007; RRID:CVCL_HD57 Human DLD-1 APEX1-/- This manuscript N/A Human DLD-1 DNMT1NA Scelfo et al.45 N/A Human DLD-1 DNMT3B-/- DNMT1NA Scelfo et al.45 N/A Human PEO1 BRCA2Y1655X Panzarino et al.41 N/A Human C4-2 PEO1 BRCA2WT Panzarino et al.41 N/A Experimental models: Organisms/strains Xenopus laevis females Nasco LM00535MX Xenopus laevis males Nasco LM00715MX Oligonucleotides siAPOBEC3B Horizon Cat#L-017432-00-0005 siBRCA2 Horizon Cat#L-003462-00-0010 siHMCES Sigma Cat#EHU036741-5UG siMRE11A Horizon Cat#L-009271-00-0010 siPRIMPOL Horizon Cat#L-016804-02-0005 siSMUG1 Horizon Cat#L-012838-01-0010 siTDG Horizon Cat#L-003780-01-0010 siTET2 Horizon Cat#L-013776-03-0010 Universal control siRNA Sigma-Aldrich Cat#SIC001 siRAD51 Horizon Cat#L-003530-00-0010 Recombinant DNA 6His-pET-SMUG1 This manuscript N/A pGEX-6P-1-APE1 This manuscript N/A Mre11 BACMID vector pTP2620 Bhaskara et al.33 Addgene Cat#113311; RRID:Addgene_113311 Rad50 BACMID vector pTP813 Bhaskara et al.33 Addgene Cat#113308; RRID:Addgene_113308 RAD51 Appleby et al.23 N/A (Continued on next page) Molecular Cell 84, 3026–3043.e1–e11, August 22, 2024 e2

Techniques: Isolation, Labeling, Generated

Journal: Molecular cell

Article Title: RAD51 protects abasic sites to prevent replication fork breakage.

doi: 10.1016/j.molcel.2024.07.004

Figure Lengend Snippet: Figure 7. Model for BRCA2- and RAD51- mediated protection against AP site- induced fork breakage (A) Unrepaired AP sites stall nascent DNA syn- thesis. Restart by PRIMPOL leads to ssDNA gaps. RAD51 binds to unrepaired AP sites. Stabilized by BRCA2, RAD51 NPFs suppress AP sites cleavage by MRE11 and restrict further SMUG1 driven AP- site accumulation. Gap sealing is facilitated by POLq and, possibly, TLS polymerases bypassing AP sites. (B) Without BRCA2/RAD51, unrestricted SMUG1 access to ssDNA promotes AP sites and abasic gap accumulation. MRE11 extends ssDNA gaps and cleaves unprotected AP sites within ssDNA, especially in the absence of POLq, causing repli- cation fork breakage. (C) APOBEC3B-dependent deamination of 5hmdC or 5mdC leads to transient 5hmdU formation, directly or indirectly. SMUG1 mediated removal of 5hmU promotes AP sites accumulation in ssDNA. (D) TET2-dependent hydroxylation of 5mdC fol- lowed by TDG-mediated removal of the 5hmdC derivatives promotes the formation of AP sites in dsDNA repaired by BER.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER xAPE1 protein This manuscript N/A GST-BRC4 Hashimoto et al.14 N/A Geminin Aze et al.85 N/A POLq protein full length Mann et al.15 N/A Critical commercial assays CellTiter-Glo Luminescent Cell Viability Assay Promega Cat#G7571 Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye, Alexa Fluor 647 dye ThermoFisher Cat#C10337; CHEBI:52661 Cat#C10340; CRISPR Gene Knockout Kit v2 targeting APEX1 Sinthego N/A DNeasy Blood & Tissue Kit Qiagen Cat#69504; Lipofectamine CRISPRMAX Transfection Reagent ThermoFisher Cat#CMAX00003; Nucleostain- DNA Damage Quantification Kit -AP Site Counting Dojindo Cat#DK02-12; Qubit dsDNA HS Assay Kits ThermoFisher Scientific Cat#Q32854; RNAiMax ThermoFisher Cat#13778150; Deposited data Atomic Coordinates and CryoEM maps Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) PDB: 8RCD, 8RCF;EMDB: EMD-19050, EMD-19051 Images and graphs Mendeley Mendeley Data: https://doi.org/10.17632/2gjvrzddv7.1 Experimental models: Cell lines Human DLD-1 wild-type Horizon Cat#HD PAR-008; RRID:CVCL_0248 Human DLD-1 BRCA2-/- Horizon Cat#HD 105-007; RRID:CVCL_HD57 Human DLD-1 APEX1-/- This manuscript N/A Human DLD-1 DNMT1NA Scelfo et al.45 N/A Human DLD-1 DNMT3B-/- DNMT1NA Scelfo et al.45 N/A Human PEO1 BRCA2Y1655X Panzarino et al.41 N/A Human C4-2 PEO1 BRCA2WT Panzarino et al.41 N/A Experimental models: Organisms/strains Xenopus laevis females Nasco LM00535MX Xenopus laevis males Nasco LM00715MX Oligonucleotides siAPOBEC3B Horizon Cat#L-017432-00-0005 siBRCA2 Horizon Cat#L-003462-00-0010 siHMCES Sigma Cat#EHU036741-5UG siMRE11A Horizon Cat#L-009271-00-0010 siPRIMPOL Horizon Cat#L-016804-02-0005 siSMUG1 Horizon Cat#L-012838-01-0010 siTDG Horizon Cat#L-003780-01-0010 siTET2 Horizon Cat#L-013776-03-0010 Universal control siRNA Sigma-Aldrich Cat#SIC001 siRAD51 Horizon Cat#L-003530-00-0010 Recombinant DNA 6His-pET-SMUG1 This manuscript N/A pGEX-6P-1-APE1 This manuscript N/A Mre11 BACMID vector pTP2620 Bhaskara et al.33 Addgene Cat#113311; RRID:Addgene_113311 Rad50 BACMID vector pTP813 Bhaskara et al.33 Addgene Cat#113308; RRID:Addgene_113308 RAD51 Appleby et al.23 N/A (Continued on next page) Molecular Cell 84, 3026–3043.e1–e11, August 22, 2024 e2

Techniques: