Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 3. Microscopic image and growth curve of HEK293 WT and RH2A-KO cells.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques:

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 5. Expression of RH2A.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Expressing

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 6. Expression of RH2A variants with AGS- causing mutation in RH2A-KO cells.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Expressing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases

doi: 10.1093/nar/gkaa948

Figure Lengend Snippet: Human DDX3X has RNaseH2-like activity. ( A ) Time course of DDX3X (lanes 1–5) and RNaseH2 (lanes 6–10) digestions of Substrate * D 39 R 1 D 15 : D 55 . ( B ) Apparent digestion rates for DDX3X and RNaseH2 enzymes on Substrate * D 39 R 1 D 15 : D 55 . Values are the means of three independent estimates ± S.D. ( C ) Digestion by DDX3X (lanes 2 and 5) and RNaseH2 (lanes 3 and 6) on Substrate * D 19 R 1 D 4 : D 24 and Substrate * D 18 R 1 D 5 : D 24 respectively. Lanes 1 and 4: Substrates * D 19 R 1 D 4 : D 24 and * D 18 R 1 D 5 : D 24 alone, respectively. ( D ) Substrate * D 19 R 4 D 18 : D 41 in the presence of RNaseH2 (lanes 4–6) or DDX3X (lanes 7–9). Lanes 1–2 control reactions with RNaseH2 and Substrate * D 19 R 1 D 4 : D 24 . Lane 3 Substrate * D 19 R 4 D 18 : D 41 alone. Lanes 10, 11 oligonucleotide size markers of defined lengths, used to better identify the different digestion products. ( E ) Time course of DDX3X digestion of Substrate * D 39 R 1 D 15 : D 55 (lanes 1–5) and Substrate * D 39 R 1 D 15 : D 39 8oxoG 1 D 15 (lanes 7–11). Control reactions with RNaseH2 of Substrate * D 39 R 1 D 15 : D 55 (lane 6) and Substrate * D 39 R 1 D 15 : D 39 8oxoG 1 D 15 (lane 12) respectively.

Article Snippet: Then, 1 μg of genomic DNA was digested with 1 U of E. coli RNaseH2 (BioLabs, Euroclone) or with 50 nM human recombinant RNaseH2 and the reaction was led in a dry heating block (Eppendorf ® Thermomixer ® ) at 37°C with constant shaking at 550 rpm for 2.5 h. Reaction was stopped on ice for 30 min and nick translation reaction started trough the addition of 1 U of E. coli DNA pol I (Biolabs, Euroclone) and 2 μl of Atto647N-dUTP pH 7.5 NT Labelling Kit (Jena Biosciences, Löbstedter, Germany) at 16°C for 1 h. Finally, the reaction was stopped by addition of standard gel loading buffer and loaded on a 1% agarose gel at 100 V for 2 h in TBE buffer.

Techniques: Activity Assay, Control

Journal: Nucleic Acids Research

Article Title: Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases

doi: 10.1093/nar/gkaa948

Figure Lengend Snippet: DDX3X RNaseH2-like activity is not dependent by its helicase activity and is inhibited by ATP. ( A ) Titration of DDX3X wild type (lanes 2–6) in helicase reaction with a dsRNA substrate 18/38mer with a 20 nt 3'-ss overhang (Substrate *R 18 : R 38 ). Lane 1: Substrate *R 18 : R 38 alone. Lane 7: 18mer single strand. ( B ) Native PAGE of the products of DDX3X helicase reaction with Substrate * D 18 R 1 D 5 : D 24 in the presence (lanes 1–5) or in the absence (lanes 7–11) of ATP. Lanes 6, 12: ss24mer as marker. ( C ) Dependence of the DDX3X helicase activity from the enzyme concentration. ( D ). Dependence of the DDX3X nuclease activity from the enzyme concentration. ( E ) Inhibition of DDX3X nuclease activity by ATP. All values are the means of three independent estimates ± S.D.

Article Snippet: Then, 1 μg of genomic DNA was digested with 1 U of E. coli RNaseH2 (BioLabs, Euroclone) or with 50 nM human recombinant RNaseH2 and the reaction was led in a dry heating block (Eppendorf ® Thermomixer ® ) at 37°C with constant shaking at 550 rpm for 2.5 h. Reaction was stopped on ice for 30 min and nick translation reaction started trough the addition of 1 U of E. coli DNA pol I (Biolabs, Euroclone) and 2 μl of Atto647N-dUTP pH 7.5 NT Labelling Kit (Jena Biosciences, Löbstedter, Germany) at 16°C for 1 h. Finally, the reaction was stopped by addition of standard gel loading buffer and loaded on a 1% agarose gel at 100 V for 2 h in TBE buffer.

Techniques: Activity Assay, Titration, Clear Native PAGE, Marker, Concentration Assay, Inhibition

Journal: Nucleic Acids Research

Article Title: Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases

doi: 10.1093/nar/gkaa948

Figure Lengend Snippet: Identification of DDX3X residues important for its RNaseH2-like activity. ( A ) Sequence alignment showing amino acid (aa) similarities between the conserved -DEAD- (box II) helicase motif of DDX3X and the catalytic site of human RNaseH2. Red letters indicate catalytically important residues. Green shading indicated identical and cyan shading similar aa. The numbers of the aa residues corresponding to each selected region are boxed. ( B ) Structure comparison between human DDX3X in open conformation (PDB ID: 5e7I) on the left and mouse RNaseH2 (PDB ID: 3KIO) of the region surrounding Arg351 of DDX3X (left) and Arg38 of RNaseH2 (right), showing the hydrogen bond formed by these residues with the RNA substrate. Arg351 in DDX3X and Arg38 in RNase H2 were similarly positioned within these regions. ( C ) Graphical representation of all the DDX3X mutants and DDX5 protein. N and C indicate the N-terminal and C-terminal of the proteins respectively. Boxes represent all the conserved motifs of DEAD-box proteins (with the addition of DDX3 unique motif). Red letters indicate single aa mutations. ( D ) Nuclease activities of the DDX3X mutants and DDX5 protein relative to DDX3X wild type (taken as 100%). All catalytic activities were assessed on Substrate * D 39 R 1 D 15 : D 55 . Values are the mean of four independent experiments ± S.D. All proteins were tested at 2 μM. ( E ) Summary table of the catalytic activities shown in panel D.

Article Snippet: Then, 1 μg of genomic DNA was digested with 1 U of E. coli RNaseH2 (BioLabs, Euroclone) or with 50 nM human recombinant RNaseH2 and the reaction was led in a dry heating block (Eppendorf ® Thermomixer ® ) at 37°C with constant shaking at 550 rpm for 2.5 h. Reaction was stopped on ice for 30 min and nick translation reaction started trough the addition of 1 U of E. coli DNA pol I (Biolabs, Euroclone) and 2 μl of Atto647N-dUTP pH 7.5 NT Labelling Kit (Jena Biosciences, Löbstedter, Germany) at 16°C for 1 h. Finally, the reaction was stopped by addition of standard gel loading buffer and loaded on a 1% agarose gel at 100 V for 2 h in TBE buffer.

Techniques: Activity Assay, Sequencing, Comparison

Journal: Nucleic Acids Research

Article Title: Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases

doi: 10.1093/nar/gkaa948

Figure Lengend Snippet: Different DNA polymerases support full ribonucleotide excision repair together with RNaseH2. ( A ) RER reactions in the presence of RNaseH2, Pol β and dNTPs alone (lane 3), or with DNA ligase 1 and Fen-1 (lanes 4–5). Lane 5: re-digestion by RNaseH2 of the ligated products after heat inactivation of the reaction. RER reactions in the presence of RNaseH2, Pol δ, PCNA and dNTPs (lane 6), or with added DNA ligase 1 and Fen-1 (lanes 7–8). Lane 8: re-digestion by RNaseH2 as in Lane 5. Lane 1: Substrate * D 39 R 1 D 15 : D 55 alone. ( B ) RER reactions in the presence of RNaseH2, Pol β, dNTPs, Fen-1 and DNA ligase 1 (lanes 2–5). RER reactions in the presence of RNaseH2, Pol δ, PCNA and dNTPs (lanes 6–8), DNA ligase 1 and Fen-1 (lanes 7–8). Lanes 5 and 8: re-digestion by RNaseH2. Lane 1: Substrate * D 19 R 4 D 18 : D 41 alone. ( C ) RER reactions in the presence of RNaseH2 (lanes 2–3), Pol λ, dNTPs (lanes 4–5), Fen-1 and DNA ligase 1 (lanes 6–7). Lanes 3, 5 and 7: re-digestion by RNaseH2. Lane 1: Substrate * D 39 R 1 D 15 : D 55 alone. ( D ) Lane 1: Substrate * D 19 R 4 D 18 : D 41 alone. RER reactions in the presence of RNaseH2 (lanes 2–3), Pol λ, dNTPs (lanes 4–5), Fen-1 and DNA ligase 1 (lanes 6–7). Lanes 3, 5 and 7: re-digestion by RNaseH2.

Article Snippet: Then, 1 μg of genomic DNA was digested with 1 U of E. coli RNaseH2 (BioLabs, Euroclone) or with 50 nM human recombinant RNaseH2 and the reaction was led in a dry heating block (Eppendorf ® Thermomixer ® ) at 37°C with constant shaking at 550 rpm for 2.5 h. Reaction was stopped on ice for 30 min and nick translation reaction started trough the addition of 1 U of E. coli DNA pol I (Biolabs, Euroclone) and 2 μl of Atto647N-dUTP pH 7.5 NT Labelling Kit (Jena Biosciences, Löbstedter, Germany) at 16°C for 1 h. Finally, the reaction was stopped by addition of standard gel loading buffer and loaded on a 1% agarose gel at 100 V for 2 h in TBE buffer.

Techniques:

Journal: Nucleic Acids Research

Article Title: Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases

doi: 10.1093/nar/gkaa948

Figure Lengend Snippet: A role of DDX3X in ribonucleotide excision repair. ( A ) RER reactions in the presence of DDX3X (lanes 2–3), Pol β, dNTPs (lanes 4–5), Fen-1 and DNA ligase 1 (lanes 6–7). Lanes 3, 5, 7: re-digestion by RNaseH2 of the heat inactivated reactions. RER reactions in the presence of DDX3X, Pol δ, PCNA, dNTPs (lanes 8–9), Fen-1 and DNA ligase 1 (lanes 10–11). Lanes 9 and 11: re-digestion by RNaseH2. Lane 1: Substrate * D 39 R 1 D 15 : D 55 alone. ( B ) RER reactions in the presence of DDX3X (lanes 2–3), Pol λ, dNTPs (lanes 4–5), Fen-1 and DNA ligase 1 (lanes 6–7). Lanes 3, 5 and 7: re-digestion by RNaseH2. Lane 1: Substrate * D 39 R 1 D 15 : D 55 alone. ( C ) Western blot for DDX3X and β-actin. L, molecular weight markers. NSC, non-silencing vector control cells; shDDX3X, stable DDX3X-silenced cells. ( D ) Quantification of genomic rNMPs by the riboassay. NSC untreated cells signal was set as 1 fluorescence arbitrary unit. Different treatments were: untreated genomic DNA (–), genomic DNA treated with 1 U of E. coli RNaseH2 or with 50 nM of human recombinant RNaseH2 (+). Data represent mean ± SEM of at least four independent experiments. ( E ) Western blot for vinculin, DDX3X and RNaseH2A. L, molecular weight markers. siCNT, non-silencing smart-pool siRNAs; siDDX3X, DDX3X-silenced cells, siRH2A, RNaseH2A-silenced cells. ( F ) Quantification of genomic rNMPs by the riboassay. siCNT (control) cells signal was set as 1 fluorescence arbitrary unit. As above, different treatments were: untreated genomic DNA (–) and genomic DNA treated with 50 nM of human recombinant RNaseH2 (+). Data represent mean ± SEM of at least four independent experiments.

Article Snippet: Then, 1 μg of genomic DNA was digested with 1 U of E. coli RNaseH2 (BioLabs, Euroclone) or with 50 nM human recombinant RNaseH2 and the reaction was led in a dry heating block (Eppendorf ® Thermomixer ® ) at 37°C with constant shaking at 550 rpm for 2.5 h. Reaction was stopped on ice for 30 min and nick translation reaction started trough the addition of 1 U of E. coli DNA pol I (Biolabs, Euroclone) and 2 μl of Atto647N-dUTP pH 7.5 NT Labelling Kit (Jena Biosciences, Löbstedter, Germany) at 16°C for 1 h. Finally, the reaction was stopped by addition of standard gel loading buffer and loaded on a 1% agarose gel at 100 V for 2 h in TBE buffer.

Techniques: Western Blot, Molecular Weight, Plasmid Preparation, Control, Fluorescence, Recombinant