Journal: Nature Communications

Article Title: RPA shields inherited DNA lesions for post-mitotic DNA synthesis

doi: 10.1038/s41467-021-23806-5

Figure Lengend Snippet: a Asynchronously growing U-2 OS cells were treated with aphidicolin (APH, 0.2 μM), ATR inhibitor (ATRi, 1μM), or APH + ATRi for 24h as indicated. 5-Ethynyl-2′-deoxyuridine (EdU) was added 20 min before fixation. Cells were stained for 53BP1 and RPA70, and cell cycle staging was performed based on DAPI and EdU by QIBC (see Supplementary Fig. and Methods section for details). Depicted are cell cycle-resolved scatter plots, in which G1 cells are labeled in blue, S phase cells in light red, and G2/M cells in dark red. Nuclear 53BP1 foci are on the y -axis, DNA content is on the x -axis. Each dot represents a single cell with its foci count. A rescaled version is shown on the right with a focus on cells in G1. Color-code as indicated. At least 1000 cells per condition were analyzed. b For the same cell populations depicted in (a) RPA foci were quantified and are shown in a cell cycle-resolved manner. c Average RPA foci counts in G1 in the different treatment conditions from n = 3 independent samples with n 1 = 499, n 2 = 495, n 3 = 476 (Control), n 1 = 367, n 2 = 345, n 3 = 278 (APH), n 1 = 944, n 2 = 1046, n 3 = 968 (ATRi), n 1 = 959, n 2 = 1042, n 3 = 1044 (APH + ATRi) cells in G1 per sample. Individual average values and means ± SD are shown. P -values were determined by two-tailed unpaired t -test; * p < 0.05 (exact p -values are p = 0.0291 and p = 0.0392, respectively), ** p < 0.01 (exact p -value is p = 0.0074). d Representative images of individual cells in G1 are shown. The upper row shows the EdU and DAPI signals used by QIBC to identify G1 cells (see Supplementary Fig. ). The lower rows show G1 cells with 53BP1 and RPA foci. Scale bar: 10 μm. A. U., arbitrary units. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used in this study: RPA70 (rabbit, Abcam ab79398, 1:500), RPA32 (mouse, Abcam ab2175, 1:500), TRF2 (rabbit, Biolegend NB110-57130, 1:500), TRF2 (mouse, Abcam ab13579, 1:500), 53BP1 (mouse, Upstate MAB3802, 1:500), 53BP1 (rabbit, NB100-304 Novus, 1:1000), Cyclin A (rabbit, Santa Cruz sc-751, 1:100), Cyclin A (mouse, Abcam ab16726, 1:200), PML PG-M3 (mouse, Santa Cruz sc-966, 1:100), H2AX Phospho S139 (mouse, Biologend 613401, 1:500).

Techniques: Staining, Labeling, Control, Two Tailed Test

Journal: Nature Communications

Article Title: RPA shields inherited DNA lesions for post-mitotic DNA synthesis

doi: 10.1038/s41467-021-23806-5

Figure Lengend Snippet: a General scheme of the experimental design to deplete RPA in G1 cells. Briefly, U-2 OS cells were arrested in G2/M by nocodazole, a mitotic shake-off was performed and cells were released in fresh medium for 4 h, allowing them to pass through mitosis. RPA depletion was then performed for 20 h in presence of CDKi RO-3306 (to block remaining S/G2 cells from cell division) and EdU (to mark cells entering S-phase and to be able to discriminate them from G1 cells), in order to assess telomere damage in G1. b Telomeric DNA damage in G1 as measured by 53BP1 co-localization with TRF2 in n = 72 G1 cells. Horizontal lines indicate means ± SD of the depicted single cell data; two-sided Mann–Whitney U test was performed. * p < 0.05 (exact p -value is p = 0.0469). c Percentage of G1 cells with damaged telomeres upon RPA depletion from n = 3 independent samples with n = 24 cells per sample. Control cells are in gray, RPA-depleted cells in red. Individual values and means ± SD are shown. P -values were determined by two-tailed unpaired t -test ** p < 0.01 (exact p -value is p = 0.0031). d Representative images of G1 cells with telomeric DNA damage upon RPA depletion in G1. Scale bar 10 μm. A. U., arbitrary units. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used in this study: RPA70 (rabbit, Abcam ab79398, 1:500), RPA32 (mouse, Abcam ab2175, 1:500), TRF2 (rabbit, Biolegend NB110-57130, 1:500), TRF2 (mouse, Abcam ab13579, 1:500), 53BP1 (mouse, Upstate MAB3802, 1:500), 53BP1 (rabbit, NB100-304 Novus, 1:1000), Cyclin A (rabbit, Santa Cruz sc-751, 1:100), Cyclin A (mouse, Abcam ab16726, 1:200), PML PG-M3 (mouse, Santa Cruz sc-966, 1:100), H2AX Phospho S139 (mouse, Biologend 613401, 1:500).

Techniques: Blocking Assay, MANN-WHITNEY, Control, Two Tailed Test

Journal: Nature Communications

Article Title: RPA shields inherited DNA lesions for post-mitotic DNA synthesis

doi: 10.1038/s41467-021-23806-5

Figure Lengend Snippet: a Under-replicated DNA, e.g., at common fragile sites (CFS), is transmitted from S/G2 to mitosis, giving rise to 53BP1 nuclear bodies in G1. b ssDNA lesions at chromosome ends, e.g., ALT intermediates, are transmitted from S/G2 to mitosis and to G1, where they are protected by RPA to allow for post-mitotic DNA synthesis (post-MiDAS). Replication stress in the previous cell cycle and/or incomplete MiDAS exacerbate heritable telomere lesions, and failure to maintain telomere integrity in the subsequent G1 phase, e.g., by loss of RPA protection, leads to telomere damage and elevated association of 53BP1 with broken telomeres.

Article Snippet: The following primary antibodies were used in this study: RPA70 (rabbit, Abcam ab79398, 1:500), RPA32 (mouse, Abcam ab2175, 1:500), TRF2 (rabbit, Biolegend NB110-57130, 1:500), TRF2 (mouse, Abcam ab13579, 1:500), 53BP1 (mouse, Upstate MAB3802, 1:500), 53BP1 (rabbit, NB100-304 Novus, 1:1000), Cyclin A (rabbit, Santa Cruz sc-751, 1:100), Cyclin A (mouse, Abcam ab16726, 1:200), PML PG-M3 (mouse, Santa Cruz sc-966, 1:100), H2AX Phospho S139 (mouse, Biologend 613401, 1:500).

Techniques: DNA Synthesis

Journal: Nature

Article Title: Polθ is phosphorylated by PLK1 to repair double-strand breaks in mitosis

doi: 10.1038/s41586-023-06506-6

Figure Lengend Snippet: a , Representative images and quantification of GFP-Polθ foci formation in cells expressing wild-type (WT) or mutant (AA) 53BP1 upon irradiation in mitosis. 30 cells were analyzed among 3 replicates. Data shows mean +/− S.E.M. Fisher’s exact test was performed to determine significance. b , Representative images and quantification of Polθ foci colocalization with PLK1 in mitosis following indicated treatment. Experiments were repeated twice with similar results. c , d , Immunoblot analysis following immunoprecipitation of GFP-tagged Polθ in asynchronous or mitotic cells upon indicated treatment. Representative experiments are shown; experiments were repeated three times with similar results. e , Representative in vitro phosphorylation assay with the Polθ fragment (E1424 to Q1503) incubated with recombinant PLK1 enzyme. Phosphorylation signals were revealed using Gel Stain and immunoblot analyses (MPM2 antibody). Cdc25 phosphorylation by PLK1 is used as a control. Representative experiments are shown; experiments were repeated twice with similar results. f , Representative in vitro kinase assay with Polθ incubated with recombinant PLK1 enzyme and 32 P-labeled ATP. Representative experiment is shown; experiment was repeated three times with similar results.

Article Snippet: For immunofluorescence, antibodies were purchased from Institut Curie antibody platform (HA), Immunovision (CREST HCT-0100), Chromotek (mNeonGreen 32F6), Novus Biologicals (FANCD2 NB100-182), Bethyl Laboratories (H2A.X (Ser139) A300-081A; TOPBP1 A300-111), Abcam (MDC1 ab50003; 53BP1 ab21083), Millipore (H2A.X (Ser139) 05-636; phospho-Histone H3 (Ser10) 06-570; 53BP1 MAB3802), and Santa Cruz Biotechnology (cyclin A sc-271682; PCNA sc-56, PLK1 sc-5504).

Techniques: Expressing, Mutagenesis, Irradiation, Western Blot, Immunoprecipitation, In Vitro, Phospho-proteomics, Incubation, Recombinant, Staining, Control, Kinase Assay, Labeling

Journal: Nature

Article Title: Polθ is phosphorylated by PLK1 to repair double-strand breaks in mitosis

doi: 10.1038/s41586-023-06506-6

Figure Lengend Snippet: a , b , Representative images of Polθ nuclear bodies (Polθ NBs) and 53BP1 nuclear bodies (53BP1 NBs) in cyclin A-negative (G1) cells. c , Size and intensity of Polθ foci in indicated cell cycle phases. From left to right, n = 98, 15, 86, 41. d , Trajectories of individual Polθ NBs in BRCA2 −/− RPE-1 cells blocked in G1 (Palbociclib treatment). Representative images of Polθ NBs from formation at mitotic exit (flattening) to dissolution in G1 (PCNA-negative cells) in BRCA2 −/− cells. e , Representative images and quantification of 53BP1 NBs in WT and POLQ −/− cells upon indicated treatment. From left to right, n = 45, 46, 53, 47. f , Representative images and quantification of 53BP1 NBs in BRCA2 −/− cells following Polθ depletion (IAA treatment). From left to right, n = 43, 29. g , h , Representative images and quantification of Polθ NBs in FANCD2-negative (G1) cells upon indicated treatment. i , Immunoblot analysis at indicated time points upon Polθ degradation (IAA treatment). In (g), from left to right, n = 561, 448 (4, 2 replicates). j , Quantification of micronuclei (MN) positive for γH2AX and/or CREST (centromere marker) in BRCA2 −/− cells following Polθ degradation (IAA treatment). From left to right, n = 75, 57; among two replicates. k , Quantification of Polθ foci in micronuclei (MN) in indicated cell lines. Scale bars represent 5 μm, except where indicated. Data represents three biological replicates, except where indicated. Data shows mean +/− S.E.M., except box plots (c) showing median with quartiles. For (c,f,j) two-tailed Mann-Whitney test; for (e) unpaired t-test was performed.

Article Snippet: For immunofluorescence, antibodies were purchased from Institut Curie antibody platform (HA), Immunovision (CREST HCT-0100), Chromotek (mNeonGreen 32F6), Novus Biologicals (FANCD2 NB100-182), Bethyl Laboratories (H2A.X (Ser139) A300-081A; TOPBP1 A300-111), Abcam (MDC1 ab50003; 53BP1 ab21083), Millipore (H2A.X (Ser139) 05-636; phospho-Histone H3 (Ser10) 06-570; 53BP1 MAB3802), and Santa Cruz Biotechnology (cyclin A sc-271682; PCNA sc-56, PLK1 sc-5504).

Techniques: Dissolution, Western Blot, Marker, Two Tailed Test, MANN-WHITNEY