circular single stranded dna substrate  (New England Biolabs)

Journal: eLife

Article Title: A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity

doi: 10.7554/eLife.45393

Figure Lengend Snippet: ( A ) Purified recombinant ribonucleases J1, J2, and a catalytically-dead variant of J1 bearing H74A and H76A mutations (dJ1). Proteins were resolved by SDS-PAGE and visualized using Coomassie G-250 staining. ( B ) RNA and DNA substrates used in nuclease assays. ( C ) Ribonuclease activities of RNases J1 and J2. The 5’-end labelled 31-nucleotide RNA substrate was combined with 0.5 pmols of indicated enzyme(s). The reaction mixture was incubated at 37°C for increasing time points (0, 5, 10, or 20 min.) RNAs were resolved using denaturing urea-PAGE. ( D ) Quantification of ribonuclease activity. Shown is the average fraction RNA cleaved (±S.D.) of three independent trials (see ). ( E ) Deoxyribonuclease activities of RNases J1 and J2 on linear single-stranded DNA. The 5’-end labelled 60-nucleotide DNA substrate was combined with 0.5 pmols of indicated enzyme(s). The reaction mixture was incubated at 37°C for increasing time points (0, 2, 5, or 10 min.) DNAs were resolved using denaturing urea-PAGE. ( F ) Quantification of deoxyribonuclease activity on single-stranded DNA. The average fraction DNA cleaved (±S.D.) from three independent trials is shown (see ). ( G ) Deoxyribonuclease activities of RNases J1 and J2 on double-stranded DNA substrates. The indicated enzymes (two pmols) were combined with 0.1 μg pUC19 DNA that was supercoiled or linearized by PstI digestion. The reaction mixture was incubated at 37°C for 1 hr and DNA was resolved on an agarose gel. ( H ) Deoxyribonuclease activities of RNases J1 and J2 on single-stranded circular DNA. The indicated enzyme(s) (four pmols) were combined with 0.5 μg of phage M13MP18 DNA and the reaction mixture was incubated at 37°C for increasing time points (15, 30, 60 min.) The DNA was resolved on a 0.7% agarose gel. Images in panels G and H are representatives of four independent trials. 10.7554/eLife.45393.019 Figure 5—source data 1. Accompanies . Fractions of RNA cleaved by indicated enzymes at various time points. 10.7554/eLife.45393.020 Figure 5—source data 2. Accompanies . Fractions of DNA cleaved by indicated enzymes at various time points.

Article Snippet: Circular ssDNA (M13MP18) substrate (0.5 µg, New England Biolabs, MA, USA) was combined with 4 pmol of each cellular nuclease alone and in combination, in nuclease buffer.

Techniques: Purification, Recombinant, Variant Assay, SDS Page, Staining, Incubation, Activity Assay, Agarose Gel Electrophoresis

Journal: eLife

Article Title: A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity

doi: 10.7554/eLife.45393

Figure Lengend Snippet:

Article Snippet: Circular ssDNA (M13MP18) substrate (0.5 µg, New England Biolabs, MA, USA) was combined with 4 pmol of each cellular nuclease alone and in combination, in nuclease buffer.

Techniques: Isolation, Recombinant, CRISPR, Over Expression, Purification, Sequencing, Construct, Real-time Polymerase Chain Reaction, Plasmid Preparation, DNA Purification, SYBR Green Assay, Software

Journal: Nature cell biology

Article Title: EXD2 promotes homologous recombination by facilitating DNA-end resection

doi: 10.1038/ncb3303

Figure Lengend Snippet: a) Quantification of the signal intensity of RPA foci in U2OS cells depleted for EXD2, MRE11 or both by siRNA (as indicated) at various time-points following treatment with 8Gy IR. ImageJ was used to quantify signal intensity per nucleus (using RPA as a marker of resection, DAPI staining marks the nucleus). n = 154, 155, 150, and 161 cells for siControl 0, 30, 60 and 120 min post-treatment, respectively. n = 177, 187, 189, and 173 cells for siEXD2 0, 30, 60 and 120 min post-treatment, respectively. n = 182, 167, 184, and 187 cells for siMRE11 0, 30, 60 and 120 min post-treatment, respectively. n = 214, 182, 150 and 163 cells for siEXD2/siMRE11 0, 30, 60 and 120 min post-treatment, respectively. In all cases cells were pooled from three independent experiments. Error bars represent +/− SEM. Statistical significance was determined using the Mann-Whitney test. b) EXD2 stimulates MRN complex–dependent nuclease activity. MRN complex (MRE11-RAD50-NBS1) (50 nM) was incubated with PhiX174 substrate DNA in the presence or absence of EXD2 WT (K76 - V564) or corresponding mutant protein (350 nM). Reaction products were resolved on agarose gel, stained with SYBR Gold and visualized. c) Quantification of the percentage of intact DNA from (b) representing DNA degradation efficiency. The percentage of intact DNA was obtained relative to the no-protein control sample. Error bars represent +/− SEM, n= 4 independent experiments, student’s t-test was used to determine statistical significance. d) and e) 5′ radiolabeled dsDNA substrate (10 nM molecules) containing a nick (d) or a 1 nucleotide gap (e) was incubated for the indicated amounts of time with EXD2 WT (K76 - V564) or EXD2 (K76 - V564) D108A E110A protein (100 nM). Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. These experiments were carried out three times independently. f) Quantification of the efficiency of DNA degradation from (d) and (e). Error bars represent +/− SEM, n = 3 independent experiments.

Article Snippet: The PhiX174 circular single-stranded substrate (30 μM nucleotides) from New England Biolabs was incubated with MRN complex (50 nM) in the presence or absence of His-EXD2 (K76-S589) (350 nM) or corresponding mutant protein in buffer containing (20 mM HEPES-KOH, pH 7.5, 50 mM KCl, 0.5 mM DTT, 5 mM MnCl 2 , 0.05% Triton-X, 0.1 mg ml −1 BSA, 5% glycerol, 1 mM ATP).

Techniques: Marker, Staining, MANN-WHITNEY, Activity Assay, Incubation, Mutagenesis, Agarose Gel Electrophoresis, Control