Journal: The Journal of Cell Biology

Article Title: Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells

doi: 10.1083/jcb.201406099

Figure Lengend Snippet: RAD51 is present at forks upon mild genotoxic stress and modulates fork progression and integrity. (A) Linear regression analysis shows strict direct correlation (P < 0.0001) between accumulation of ssDNA at the fork (median values of ssDNA regions at the junction) and frequency of fork reversal. Results from two independent experiments are displayed for untreated U2OS cells and for each genotoxic treatment. (B) HEK293T cells were EdU-labeled as indicated in Fig. S5 C and treated with sublethal doses of genotoxic drugs (0.5 mM HU, 200 nM MMC, or 25 nM CPT). Proteins and relative posttranslational modifications associated with replication forks were isolated by iPOND procedure and detected with the indicated antibodies. The thymidine (Thy; 10 µM) chase experiment is used to discriminate proteins associated with chromatin behind replicating forks. In the control (Ctrl) experiment, the click reaction is performed using DMSO instead of biotin azide. 1 µM CPT treatment is used as positive control to induce high replication stress and DSBs. (C) Immunofluorescence staining for U2OS cells grown on coverslips and treated with the indicated drugs for 1 h. Red staining, RAD51; green staining, EdU; blue, DAPI. Bar, 15 µM. (D) DNA fiber spreading. Statistical analysis of IdU replicated track length in U2OS cells, comparing not treated (NT) conditions with the indicated treatments. U2OS cells were transfected with siRNA against luciferase (siLuc) or RAD51 (siRAD51) 24 h before CldU or IdU labeling. At least 100 tracks were scored per sample. Horizontal lines represent the median value, and boxes and whiskers indicate 10–90th percentiles. Statistical analysis: one-way ANOVA; ns, not significant; ***, P ≤ 0.001. (E) PFGE analysis for DNA breakage detection in untreated U2OS cells and upon 1-h treatment with indicated doses of genotoxic treatments. U2OS cells were transfected with siRNA against luciferase or RAD51 24 h before treatments. 1 µM camptothecin (CPT) treatment is used as a positive control for DSB formation. The graph shows quantitative DSB induction from three independent experiments and includes average value and standard deviations (error bars). Statistical analysis: two-way ANOVA; ns, not significant; *, P ≤ 0.05.

Article Snippet: Electrophoresis was performed for 21 h at 14°C in 0.9% (wt/vol) Pulse Field Certified Agarose (Bio-Rad Laboratories) containing Tris-borate/EDTA buffer in a PFGE apparatus (CHEF DR III; Bio-Rad Laboratories), according to the following protocol (block I: 9 h, 120° included angle, 5.5 V/cm, 30 to 18-s switch; block II: 6 h, 117° included angle, 4.5 V/cm, 18 to 9-s switch; block III: 6 h, 112° included angle, 4.0 V/cm, 9 to 5-s switch).

Techniques: Labeling, Isolation, Control, Positive Control, Immunofluorescence, Staining, Transfection, Luciferase

Journal: Nucleic Acids Research

Article Title: Disease-associated DNA2 nuclease–helicase protects cells from lethal chromosome under-replication

doi: 10.1093/nar/gkaa524

Figure Lengend Snippet: Dna2 is required for the completion of chromosome replication. ( A ) Representative PFGE experiments with the indicated strains, DNA stained by ethidium bromide. G1-synchronized cells were released into S phase in the presence of nocodazole to arrest cells prior to mitosis. At the indicated experimental stages, genomic DNA was analyzed for fully replicated (gel-resolved) chromosomes by PFGE. Gel-resolved DNA is labelled with chromosome numbers. ( B ) Southern blot analysis of the gel shown in panel A, probing for chromosome XII. ( C ) Quantification of Southern blots as shown in panel B to determine the fraction of gel-resolved chromosome XII. Data represent mean values ± SEM ( n = 3 independent experiments). ( D ) Cell-cycle progression analysis by flow cytometry of cells collected as in panel A.

Article Snippet: Plugs were treated 1 h at 37°C with 0.4 mg ml −1 Zymolyase (USBiological), overnight at 50°C with proteinase K (Eurobio) in 10 mM Tris–HCl, 50 mM EDTA, 1% N -lauroylsarcosinate (Sigma), and then washed with ddH 2 O. Genomic DNA was migrated through 1% agarose in 0.5× TBE (89 mM Tris–HCl, 89 mM boric acid, 2 mM EDTA) in a CHEF-DR II PFGE system (BioRad) maintained at 14°C, 6 V/cm, 60 s switch time for 15 h, followed by 90 s switch time for 9 h. Ethidium bromide-stained DNA was visualized using a GE Typhoon 9400 system.

Techniques: Staining, Southern Blot, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Disease-associated DNA2 nuclease–helicase protects cells from lethal chromosome under-replication

doi: 10.1093/nar/gkaa524

Figure Lengend Snippet: Transient replication arrest induces under-replication in the absence of Dna2. ( A ) Representative PFGE of the indicated strains treated with HU, DNA stained by ethidium bromide. G1-synchronized cells were released into medium containing 200 mM HU for 2 h, followed by drug wash-out and incubation in HU-free medium containing nocodazole to prevent mitosis. At the indicated stages, genomic DNA was analyzed for fully replicated (gel-resolved) chromosomes by PFGE. Gel-resolved DNA is labelled with chromosome numbers. ( B ) Cell-cycle progression analysis by flow cytometry of cells collected for the experiment shown in panel A. ( C ) Southern blot analysis of a gel obtained as in panel A, probing for chromosome XII. ( D ) Quantification of Southern blots as shown in panel C to determine the fraction of gel-resolved chromosome XII. Data represent mean values ± SEM ( n = 3 independent experiments). ( E ) Southern blot analysis of a gel obtained as in panel A, probing for chromosome XIII. ( F ) Quantification of Southern blots as shown in panel E to determine the fraction of gel-resolved chromosome XIII. Data represent mean values ± SEM ( n = 3 independent experiments).

Article Snippet: Plugs were treated 1 h at 37°C with 0.4 mg ml −1 Zymolyase (USBiological), overnight at 50°C with proteinase K (Eurobio) in 10 mM Tris–HCl, 50 mM EDTA, 1% N -lauroylsarcosinate (Sigma), and then washed with ddH 2 O. Genomic DNA was migrated through 1% agarose in 0.5× TBE (89 mM Tris–HCl, 89 mM boric acid, 2 mM EDTA) in a CHEF-DR II PFGE system (BioRad) maintained at 14°C, 6 V/cm, 60 s switch time for 15 h, followed by 90 s switch time for 9 h. Ethidium bromide-stained DNA was visualized using a GE Typhoon 9400 system.

Techniques: Staining, Incubation, Flow Cytometry, Southern Blot

Journal: Journal of applied microbiology

Article Title: Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity.

doi: 10.1046/j.1365-2672.2003.02134.x

Figure Lengend Snippet: Fig. 1 Pulsed-field gel electrophoresis patterns of Sma I digests of genomic DNA from Lactococcus lactis subsp. lactis strains. Lanes 1–10, strains LW2004, LW2006, LW1999, LW2003, LW2005, LW2000, LW2001, LW2002, LW2515, IL1403. The pulse time used was 1–30 s

Article Snippet: DNA embedded in agarose was digested with Sma I (New England Biolabs, Beverly, MA, USA), loaded into the wells of 1% agarose gels (pulsed-field certified agarose; BioRad Laboratories, Hercules, CA, USA), and separated at 200 V for 20 h at 14 C in 0Æ5X Tris-borate buffer using a CHEF DR II PFGE apparatus and model 1000 mini chiller (Bio-Rad).

Techniques: Nucleic Acid Electrophoresis