Journal: Stem Cell Reports

Article Title: Endogenous DNA Damage Leads to p53-Independent Deficits in Replicative Fitness in Fetal Murine Fancd2 − / − Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.stemcr.2016.09.005

Figure Lengend Snippet: DNA-Damage Responses Are Induced, and the Strand Breaks Are Accumulated in FA FL HSPCs (A) Representative γH2AX foci in SCA1 + FL cells (60× objective lens; the scale bar represents 5 μm). (B) Quantification of γH2AX foci per cell (n = 3 WT and 3 KO from three litters). (C) Representative RAD51 foci in SCA1 + FL (60× objective lens; the scale bar represents 5 μm). (D) Quantification of cells positive for foci (p = 0.02, n = 6 WT and 6 KO from three litters). (E) Expression of DDR genes in SCA1 + FL cells (p = 0.08, 0.44, 0.06, 0.22, and 0.005, respectively; n = 4 WT and 4 KO from three litters). (F) Expression of selected DDR genes in ASL-sorted FL cells (p = 0.08, 0.5, 0.04 respectively; n = 3 WT and 3 KO from two litters). (G) Olive tail moment of 428 SCA1 + FL cells from seven WT animals and 288 SCA1 + FL cells from five Fancc − / − animals from four litters (p = 0.003). (H) Olive tail moment of 289 SCA1 + FL cells from five WT animals and 267 SCA1 + FL cells from five Fancd2 − / − animals from four litters (p = 0.04). (I) Representative alkaline comets of SCA1 + FL cells (20× objective lens; the scale bar represents 20 μm). Error bars reflect SEM, and asterisks indicate ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01.

Article Snippet: Cells were permeabilized with NET buffer with 0.5% Triton X-100 ( ) and stained with Hoechst (Thermo Scientific 62249) and AF488-conjugated anti-γH2AX (BioLegend, 613407), rabbit anti-mouse RAD51 (Santa Cruz Biotechnology, 8349) followed by Cy3 goat anti-rabbit IgG (Millipore, AP132C) or Cy5-conjugated anti-phospho-p38 (T180/Y182) (eBioscience, 17-9078-42).

Techniques: Expressing

Journal: International Journal of Medical Sciences

Article Title: IER5 is involved in DNA Double-Strand Breaks Repair in Association with PAPR1 in Hela Cells

doi: 10.7150/ijms.21510

Figure Lengend Snippet: IER5 regulates NHEJ-mediated DSB repair. a. Western blot indicating IER5 and 53BP1 knockdown by specific siRNAs. b. FACS analyses of NHEJ assay in Hela cells treated with IER5 and 53BP1 siRNAs. c. Quantification of the NHEJ assay. d. Western blot indicating IER5 and RAD51 knockdown in Hela cells upon treatment with specific siRNAs. e. Efficiency of HR, as analyzed by FACS. f. Quantification of the HR assay.

Article Snippet: Antibodies used in this study: anti-IER5 goat polyclonal antibody (Abcam), anti-IER5 rabbit polyclonal antibody (Santa Cruz), anti-GAPDH mouse monoclonal antibody (Zhong Shan Jin Qiao), anti-PARP1 rabbit monoclonal antibody (Santa Cruz), anti-Ku70 mouse monoclonal antibody (Abcam), anti-Ku80 rabbit monoclonal antibody (Santa Cruz), anti-FLAG M2 mouse monoclonal antibody (Sigma), anti-53BP1 rabbit polyclonal antibody (Santa Cruz), anti-RAD51 rabbit polyclonal antibody (Proteintech), anti-γH2AX mouse monoclonal antibody (Millipore), anti-Ubiquitinylated proteins mouse monoclonal antibody (Millipore), and anti-PADPR mouse monoclonal antibody (Abcam).

Techniques: Western Blot, Knockdown, NHEJ Assay

Journal: Molecular cell

Article Title: RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis.

doi: 10.1016/j.molcel.2017.05.006

Figure Lengend Snippet: Figure 1. Physical and Functional Interaction between MUS81-EME1 Endonuclease and RECQ5 (A) Time course of cleavage of 30-flap DNA substrate (6 nM) with MUS81-EME1 (ME1; 0.2 nM) in the presence or absence of RECQ5 (5 nM). (B) Quantification of (A). (C) Effect of increasing concentrations of RECQ5 on cleavage of indicated DNA substrates (6 nM) by MUS81-EME1 (0.2 nM). Reactions were incubated at 37C for 20 min. (D) Quantification of (C). (E) Physical interaction of RECQ5 with MUS81-EME1. RECQ5 was incubated for 30 min with GST-tagged MUS81-EME1 or GST alone pre-bound to glutathione Sepharose beads. Fractions of unbound (U) and bound (B) proteins were resolved by SDS-PAGE. (F) Complex formation between MUS81 and RECQ5 in human cells. Immunoprecipitation (IP) of RECQ5 from a total extract of U2OS cells was performed using rabbit polyclonal antibodies against a C-terminal (a-C-term) region of RECQ5 (675-991) and the full-length (a-FL) RECQ5, respectively. Where indicated, extracts were supplemented with ethidium bromide (EtBr; 50 mg/ml) to disrupt DNA-protein interactions. (G) Schematic representation of RECQ5 domain organization. RQC, RecQ C-terminal domain; KIX, kinase-inducible domain interacting; 51BD, RAD51-binding domain; SRI, Set2-Rpb1 interaction motif. Numbers indicate boundaries of the individual domains. (H) Mapping the MUS81-interaction domain of RECQ5. Indicated RECQ5 variants were expressed ectopically in HEK293 cells as N-terminal fusions with a 6xHis- Xpress epitope tag. Cell extracts were incubated with Ni-NTA beads, and bound proteins were analyzed by western blotting. (I) Effect of wild-type RECQ5 and RECQ5D515-568 (25 nM) on cleavage of 30-flap DNA substrate (4 nM) by MUS81-EME1 (0.2 nM). Reactions were carried out as in (A). For (B), (D), and (I), data are means of at least three independent experiments. Error bars show standard deviation (SD). See also Figure S1.

Article Snippet: The primary antibodies used for WB: FLAG mouse monoclonal (F1804, Sigma-Aldrich; 1:500 dilution), MCM7 mouse monoclonal (M7931, Sigma-Aldrich; 1:2000), MUS81 mouse monoclonal (M1445, Sigma-Aldrich; 1:2000), phospho-RECQ5 (Ser727) rabbit polyclonal (Janscak lab; 1:2000), phosho-Histone H3 (Ser10) rabbit polyclonal (06-570, Millipore; 1:1000), GFP rabbit polyclonal (ab290, Abcam; 1:2000), Omni-probe goat polyclonal (sc-499-G, Santa Cruz Biotechnology; 1:1000), RECQ5 rabbit polyclonal (Janscak lab; 1:2000), RAD51 rabbit polyclonal (sc-8349, Santa Cruz; 1:1000), b Tubulin (D-10) mouse monoclonal (sc-5274, Santa Cruz Biotechnology; 1:2000) and TFIIH rabbit polyclonal (sc293, Santa Cruz Biotechnology; 1:2000).

Techniques: Functional Assay, Incubation, SDS Page, Immunoprecipitation, Binding Assay, Western Blot, Standard Deviation

Journal: Molecular cell

Article Title: RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis.

doi: 10.1016/j.molcel.2017.05.006

Figure Lengend Snippet: Figure 5. RAD51 Filaments Inhibit MUS81-Mediated Processing of Stalled Replication Forks at CFSs in RECQ5-Deficient Cells (A) RECQ5 depletion causes excessive binding of RAD51 to CFSs. Cross-linked chromatin samples of mock- or RECQ5-depleted U2OS cells treated with APH (0.4 mM) or DMSO for 24 hr were subjected to ChIP assay with anti-RAD51 antibody or control IgG. Data represent means of three independent experiments. Error bars show SD. (B) Wstern blot analysis of extracts of U2OS T-REx cells harboring wild-type (WT), K58R, or F666A forms of siRNA-resistant RECQ5-FLAG fusion gene controlled by a doxycycline-inducible promoter. Twenty-four hours after transfection of indicated siRNAs, doxycycline (Dox) or DMSO was added with fresh medium for a further 48 hr as indicated. (C–G) Quantifications of RAD51 binding to CFSs (C), CFS expression (D), the frequency of DAPI-positive anaphase bridges (E) and micronuclei (F), and 53BP1 binding to CFSs (G) for cells in (B). Three conditions were analyzed for each cell line: siCtrl -dox (expression of endogenous RECQ5), siRECQ5 -dox (RECQ5 depletion), and siRECQ5 +dox (replacement of endogenous RECQ5 with RECQ5 variant). Data are means of three (C and G) or two (D–F) independent exper- iments. Error bars show SD in (C) and (E–G) and SEM in (D). (H) RECQ5 alleviates the inhibitory effect of RAD51 filaments on 30-flap DNA cleavage by MUS81-EME1. Fluorescently labeled 30-flap DNA substrate (6 nM) was pre-incubated for 10 min with RAD51K133R (500 nM) in the presence of ATP (2 mM) and ATP-regenerating system. Reactions were then supplemented with indicated concentrations of wild-type (WT) or mutant (K58R, F666A) forms of RECQ5 and incubated for 10 min at 37C, followed by addition of MUS81-EME1 (8 nM) and incubation for a further 20 min. (I) Quantification of (H). Data are means of at least three independent experiments. Error bars show SD. See also Figures S3, S4, S5, and S6.

Article Snippet: The primary antibodies used for WB: FLAG mouse monoclonal (F1804, Sigma-Aldrich; 1:500 dilution), MCM7 mouse monoclonal (M7931, Sigma-Aldrich; 1:2000), MUS81 mouse monoclonal (M1445, Sigma-Aldrich; 1:2000), phospho-RECQ5 (Ser727) rabbit polyclonal (Janscak lab; 1:2000), phosho-Histone H3 (Ser10) rabbit polyclonal (06-570, Millipore; 1:1000), GFP rabbit polyclonal (ab290, Abcam; 1:2000), Omni-probe goat polyclonal (sc-499-G, Santa Cruz Biotechnology; 1:1000), RECQ5 rabbit polyclonal (Janscak lab; 1:2000), RAD51 rabbit polyclonal (sc-8349, Santa Cruz; 1:1000), b Tubulin (D-10) mouse monoclonal (sc-5274, Santa Cruz Biotechnology; 1:2000) and TFIIH rabbit polyclonal (sc293, Santa Cruz Biotechnology; 1:2000).

Techniques: Binding Assay, Control, Transfection, Expressing, Variant Assay, Labeling, Incubation, Mutagenesis

Journal: Molecular cell

Article Title: RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis.

doi: 10.1016/j.molcel.2017.05.006

Figure Lengend Snippet: Figure 7. Model for the Role of RECQ5 in Facilitating CFS Cleavage by MUS81-EME1 Endonuclease during Early Mitosis Perturbation of DNA replication at late-replicating common fragile sites (CFSs) leads to the assembly of RAD51 filaments on single-stranded DNA re- gions at the stalled forks. As such, stalled forks escape G2/M checkpoint. When cells enter mitosis, SLX4 forms complex with the MUS81- EME1 endonuclease and recruits it to CFS loci. In turn, MUS81 recruits RECQ5 through direct pro- tein-protein interaction. Alternatively, RECQ5 is recruited in complex with MUS81-EME1. RECQ5 eliminates RAD51 filament on the leading strand of the stalled fork and stimulates fork cleavage by MUS81-EME1, which triggers DNA synthesis (not shown) required for correct sister chro- matid segregation. RECQ5-mediated disruption of RAD51 filaments at CFSs is driven by phosphory- lation of RECQ5 Ser727 by CDK1. RECQ5 defi- ciency leads to RAD51 persistency on replication intermediates, which consequently impedes chromosome segregation. The cell will inevitably go through an aberrant anaphase separation, leading to genomic instability.

Article Snippet: The primary antibodies used for WB: FLAG mouse monoclonal (F1804, Sigma-Aldrich; 1:500 dilution), MCM7 mouse monoclonal (M7931, Sigma-Aldrich; 1:2000), MUS81 mouse monoclonal (M1445, Sigma-Aldrich; 1:2000), phospho-RECQ5 (Ser727) rabbit polyclonal (Janscak lab; 1:2000), phosho-Histone H3 (Ser10) rabbit polyclonal (06-570, Millipore; 1:1000), GFP rabbit polyclonal (ab290, Abcam; 1:2000), Omni-probe goat polyclonal (sc-499-G, Santa Cruz Biotechnology; 1:1000), RECQ5 rabbit polyclonal (Janscak lab; 1:2000), RAD51 rabbit polyclonal (sc-8349, Santa Cruz; 1:1000), b Tubulin (D-10) mouse monoclonal (sc-5274, Santa Cruz Biotechnology; 1:2000) and TFIIH rabbit polyclonal (sc293, Santa Cruz Biotechnology; 1:2000).

Techniques: DNA Synthesis, Disruption