rrm2  (Bio-Rad)

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet: Effects of RRM2B Depletion in Hypoxia (A) Immunoprecipitation of RRM1 followed by immunoblotting for RRM2B and RRM2 in normoxia and <0.1% O 2 (18 hr). (B) dNTP levels in RKO cells treated with non-specific (siCTL) or siRRM2B and exposed to <0.1% O 2 (16 hr). (C) FACS analysis of U2OS cells treated with siCTL or siRRM2B and exposed to normoxia or <0.1% O 2 (3 hr). Cells were pulsed with bromodeoxyuridine (BrdU) (20 μM) 30 min before collection. (D) RPA32 foci in RKO RRM2B+/+ and RKO RRM2B−/− cells after exposure to <0.1% O 2 . (E) 53BP1 foci in RKO RRM2B+/+ and RKO RRM2B−/− cells exposed to normoxia or <0.1% O 2 (6 hr). (F) Representative images of 53BP1 foci in RRM2B-negative RKO cells treated with siRRM2B and exposed to normoxia or <0.1% O 2 (6 hr). Scale bar, 20 μm. (G) Colony survival assay in RKO cells treated with siCTL or siRRM2B and exposed to normoxia or <0.1% O 2 (24 hr). (H) Apoptosis detected morphologically in RKO cells treated with siCTL or siRRM2B and exposed to normoxia or <0.1% O 2 (19 hr). (I) RKO RRM2B+/+ and RKO RRM2B−/− cells were grown as xenografts in mice (n = 4 mice per each group). Where indicated, irradiation (10 Gy) was given when tumors reached ∼100 mm 3 . (J) Representative images of co-localization of cleaved caspase-3 (apoptosis) with PIMO (hypoxic areas) in RKO RRM2B+/+ or RKO RRM2B−/− xenografts. Scale bars, 50 μm. (K and L) Tumors were removed on day 28 post-implantation (from H), and the level of apoptosis was quantified in normoxic areas (PIMO negative) (K) and hypoxic areas (PIMO positive) (L). Images from three different tumors (n = 3) per group were counted. For all panels, n = 3 (biological replicates) unless otherwise stated. Data show mean ± SEM and two-tailed Student’s t test was applied, except in (D), where one-way ANOVA analysis was applied, and (I), where two-way ANOVA analysis was applied. (ns) indicates a non-significant change. See also and .

Article Snippet: Antibodies used were: RRM1 (Santa Cruz Biotechnology: sc-11733), RRM2B (Santa Cruz Biotechnology: sc-10840), RRM2 (AbD Serotec, MCA3434Z) and for the detection of RRM2 the Pierce Recombinant Protein A/G (Thermo Fisher Scientific, 21186) secondary antibody was used.

Techniques: Immunoprecipitation, Western Blot, Clonogenic Cell Survival Assay, Irradiation, Two Tailed Test

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet: RRM2B Retains Activity in Hypoxia (A) Product formation (percentage of the maximum, where maximum is the dCDP levels at 30 min in normoxia) for R1/R2B enzyme in normoxia and <0.1% O 2 . (B) dCDP (μM) in <0.1% O 2 for R1/R2B for the times indicated. Activity of R1/R2B enzyme at 37°C at 5 min in <0.1% O 2 was 19.57 nmol/min/mg RRM2B protein. Gray columns indicate the amount of dCDP formed up to 15 min in <0.1% O 2 , and red columns indicate the amount of dCDP formed after 15 min in <0.1% O 2. (C) Product formation (percentage of the maximum, where maximum is the dCDP levels at 30 min in normoxia) for R1/R2 enzyme in normoxia and <0.1% O 2 . (D) dCDP (μM) in <0.1% O 2 for R1/R2 for the times indicated. Activity of R1/R2 enzyme at 37°C at 5 min in <0.1% O 2 was 97.74 nmol/min/mg RRM2 protein. Gray columns indicate the amount of dCDP formed up to 15 min in <0.1% O 2 , and red columns indicate the amount of dCDP formed after 15 min in <0.1% O 2. (E and F) Characterization of the oxygen tunnels (T1–T3) of RRM2B (E) and RRM2 (F). (G and H) EPR spectra of the tyrosyl radical of RRM2B (G) and RRM2 (H) in normoxia and <0.1% O 2 , respectively. (I) Quantification of (G) and (H). Data present electron spins per β subunit. For all panels, n = 3 (biological replicates); for (A) and (C), data represent mean ± SEM and two-way ANOVA was applied; for (B) and (D), data represent mean ± SEM and two-tailed Student’s t test was applied; (ns) indicates non significant change. See also Figure S5 .

Article Snippet: Antibodies used were: RRM1 (Santa Cruz Biotechnology: sc-11733), RRM2B (Santa Cruz Biotechnology: sc-10840), RRM2 (AbD Serotec, MCA3434Z) and for the detection of RRM2 the Pierce Recombinant Protein A/G (Thermo Fisher Scientific, 21186) secondary antibody was used.

Techniques: Activity Assay, Two Tailed Test

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet: O 2 Residence Times around the Fe Metallocenter for RRM2B and RRM2 Proteins

Article Snippet: Antibodies used were: RRM1 (Santa Cruz Biotechnology: sc-11733), RRM2B (Santa Cruz Biotechnology: sc-10840), RRM2 (AbD Serotec, MCA3434Z) and for the detection of RRM2 the Pierce Recombinant Protein A/G (Thermo Fisher Scientific, 21186) secondary antibody was used.

Techniques:

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet: Critical Roles of K37/K151 and Y164 in RRM2B (A and B) Product formation (percentage of the maximum, where maximum is the dCDP levels at 30 min in normoxia) for K37E/K151E (A) and Y164C (B) in normoxia and <0.1% O 2 . (C) EPR spectra of the tyrosyl radical of Y164C, K37E/K151E, and Q127K (as a negative control) in normoxia and <0.1% O 2 . (D) Quantification of (C). Data present electron spins per β subunit. (E) The RRM2B phenylalanine network around Y164 and phenylalanine conformation in Y164C mutation. Distance plot reveals the effect of Y164C in F95-F197 distance. Color code: WT (black), Y164C (red). (F and G) dATP (F) and dTTP (G) levels in RKO RRM2B−/− cells transfected with CTL, WT, Y164C, or K37E/K151E and exposed to <0.1% O 2 (16 hr). (H) Immunoblot for PARP cleavage in RKO RRM2B−/− cells treated as in (F) and (G) plus Q127K and exposed to <0.1% O 2 (19 hr). (I) Apoptosis detected morphologically in RKO RRM2B−/− cells treated as in (H). (J) Schematic representation of our proposed model. Hypoxia leads to severely compromised activity of RRM2, leading to replication stress. RRM2B is then induced through the DDR pathway to maintain ongoing replication. However, insufficient dNTPs are generated by R1/R2B, and replication stress is unresolved. The importance of RRM2B activity is that while it does not resolve replication stress, it does maintain replication fork integrity and prevents the accumulation of DNA damage and loss of genome stability. For (A), n = 3; for (B), n = 4 (biological replicates) and two-way ANOVA was applied; for (C), n = 2 (biological replicates); for (F)–(I), n = 3 (biological replicates); data represent means ± SEM and two-tailed Student’s t test was applied. See also and .

Article Snippet: Antibodies used were: RRM1 (Santa Cruz Biotechnology: sc-11733), RRM2B (Santa Cruz Biotechnology: sc-10840), RRM2 (AbD Serotec, MCA3434Z) and for the detection of RRM2 the Pierce Recombinant Protein A/G (Thermo Fisher Scientific, 21186) secondary antibody was used.

Techniques: Negative Control, Mutagenesis, Transfection, Western Blot, Activity Assay, Generated, Two Tailed Test

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet:

Article Snippet: Antibodies used were: RRM1 (Santa Cruz Biotechnology: sc-11733), RRM2B (Santa Cruz Biotechnology: sc-10840), RRM2 (AbD Serotec, MCA3434Z) and for the detection of RRM2 the Pierce Recombinant Protein A/G (Thermo Fisher Scientific, 21186) secondary antibody was used.

Techniques: Transduction, Recombinant, Protease Inhibitor, SYBR Green Assay, Mutagenesis, Purification, Gel Extraction, Imaging, Sequencing, Negative Control, Real-time Polymerase Chain Reaction, Software, Expressing

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-RRM2 Clone 1E1 , Bio-Rad , Cat# MCA3434Z.

Techniques: Transduction, Recombinant, Protease Inhibitor, SYBR Green Assay, Mutagenesis, Purification, Gel Extraction, Imaging, Sequencing, Negative Control, Real-time Polymerase Chain Reaction, Software, Expressing

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet: Effects of RRM2B Depletion in Hypoxia (A) Immunoprecipitation of RRM1 followed by immunoblotting for RRM2B and RRM2 in normoxia and <0.1% O 2 (18 hr). (B) dNTP levels in RKO cells treated with non-specific (siCTL) or siRRM2B and exposed to <0.1% O 2 (16 hr). (C) FACS analysis of U2OS cells treated with siCTL or siRRM2B and exposed to normoxia or <0.1% O 2 (3 hr). Cells were pulsed with bromodeoxyuridine (BrdU) (20 μM) 30 min before collection. (D) RPA32 foci in RKO RRM2B+/+ and RKO RRM2B−/− cells after exposure to <0.1% O 2 . (E) 53BP1 foci in RKO RRM2B+/+ and RKO RRM2B−/− cells exposed to normoxia or <0.1% O 2 (6 hr). (F) Representative images of 53BP1 foci in RRM2B-negative RKO cells treated with siRRM2B and exposed to normoxia or <0.1% O 2 (6 hr). Scale bar, 20 μm. (G) Colony survival assay in RKO cells treated with siCTL or siRRM2B and exposed to normoxia or <0.1% O 2 (24 hr). (H) Apoptosis detected morphologically in RKO cells treated with siCTL or siRRM2B and exposed to normoxia or <0.1% O 2 (19 hr). (I) RKO RRM2B+/+ and RKO RRM2B−/− cells were grown as xenografts in mice (n = 4 mice per each group). Where indicated, irradiation (10 Gy) was given when tumors reached ∼100 mm 3 . (J) Representative images of co-localization of cleaved caspase-3 (apoptosis) with PIMO (hypoxic areas) in RKO RRM2B+/+ or RKO RRM2B−/− xenografts. Scale bars, 50 μm. (K and L) Tumors were removed on day 28 post-implantation (from H), and the level of apoptosis was quantified in normoxic areas (PIMO negative) (K) and hypoxic areas (PIMO positive) (L). Images from three different tumors (n = 3) per group were counted. For all panels, n = 3 (biological replicates) unless otherwise stated. Data show mean ± SEM and two-tailed Student’s t test was applied, except in (D), where one-way ANOVA analysis was applied, and (I), where two-way ANOVA analysis was applied. (ns) indicates a non-significant change. See also and .

Article Snippet: Mouse monoclonal anti-RRM2 Clone 1E1 , Bio-Rad , Cat# MCA3434Z.

Techniques: Immunoprecipitation, Western Blot, Clonogenic Cell Survival Assay, Irradiation, Two Tailed Test

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet: RRM2B Retains Activity in Hypoxia (A) Product formation (percentage of the maximum, where maximum is the dCDP levels at 30 min in normoxia) for R1/R2B enzyme in normoxia and <0.1% O 2 . (B) dCDP (μM) in <0.1% O 2 for R1/R2B for the times indicated. Activity of R1/R2B enzyme at 37°C at 5 min in <0.1% O 2 was 19.57 nmol/min/mg RRM2B protein. Gray columns indicate the amount of dCDP formed up to 15 min in <0.1% O 2 , and red columns indicate the amount of dCDP formed after 15 min in <0.1% O 2. (C) Product formation (percentage of the maximum, where maximum is the dCDP levels at 30 min in normoxia) for R1/R2 enzyme in normoxia and <0.1% O 2 . (D) dCDP (μM) in <0.1% O 2 for R1/R2 for the times indicated. Activity of R1/R2 enzyme at 37°C at 5 min in <0.1% O 2 was 97.74 nmol/min/mg RRM2 protein. Gray columns indicate the amount of dCDP formed up to 15 min in <0.1% O 2 , and red columns indicate the amount of dCDP formed after 15 min in <0.1% O 2. (E and F) Characterization of the oxygen tunnels (T1–T3) of RRM2B (E) and RRM2 (F). (G and H) EPR spectra of the tyrosyl radical of RRM2B (G) and RRM2 (H) in normoxia and <0.1% O 2 , respectively. (I) Quantification of (G) and (H). Data present electron spins per β subunit. For all panels, n = 3 (biological replicates); for (A) and (C), data represent mean ± SEM and two-way ANOVA was applied; for (B) and (D), data represent mean ± SEM and two-tailed Student’s t test was applied; (ns) indicates non significant change. See also Figure S5 .

Article Snippet: Mouse monoclonal anti-RRM2 Clone 1E1 , Bio-Rad , Cat# MCA3434Z.

Techniques: Activity Assay, Two Tailed Test

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet: O 2 Residence Times around the Fe Metallocenter for RRM2B and RRM2 Proteins

Article Snippet: Mouse monoclonal anti-RRM2 Clone 1E1 , Bio-Rad , Cat# MCA3434Z.

Techniques:

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet: Critical Roles of K37/K151 and Y164 in RRM2B (A and B) Product formation (percentage of the maximum, where maximum is the dCDP levels at 30 min in normoxia) for K37E/K151E (A) and Y164C (B) in normoxia and <0.1% O 2 . (C) EPR spectra of the tyrosyl radical of Y164C, K37E/K151E, and Q127K (as a negative control) in normoxia and <0.1% O 2 . (D) Quantification of (C). Data present electron spins per β subunit. (E) The RRM2B phenylalanine network around Y164 and phenylalanine conformation in Y164C mutation. Distance plot reveals the effect of Y164C in F95-F197 distance. Color code: WT (black), Y164C (red). (F and G) dATP (F) and dTTP (G) levels in RKO RRM2B−/− cells transfected with CTL, WT, Y164C, or K37E/K151E and exposed to <0.1% O 2 (16 hr). (H) Immunoblot for PARP cleavage in RKO RRM2B−/− cells treated as in (F) and (G) plus Q127K and exposed to <0.1% O 2 (19 hr). (I) Apoptosis detected morphologically in RKO RRM2B−/− cells treated as in (H). (J) Schematic representation of our proposed model. Hypoxia leads to severely compromised activity of RRM2, leading to replication stress. RRM2B is then induced through the DDR pathway to maintain ongoing replication. However, insufficient dNTPs are generated by R1/R2B, and replication stress is unresolved. The importance of RRM2B activity is that while it does not resolve replication stress, it does maintain replication fork integrity and prevents the accumulation of DNA damage and loss of genome stability. For (A), n = 3; for (B), n = 4 (biological replicates) and two-way ANOVA was applied; for (C), n = 2 (biological replicates); for (F)–(I), n = 3 (biological replicates); data represent means ± SEM and two-tailed Student’s t test was applied. See also and .

Article Snippet: Mouse monoclonal anti-RRM2 Clone 1E1 , Bio-Rad , Cat# MCA3434Z.

Techniques: Negative Control, Mutagenesis, Transfection, Western Blot, Activity Assay, Generated, Two Tailed Test

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-RRM2 Clone 1E1 , Bio-Rad , Cat# MCA3434Z.

Techniques: Transduction, Recombinant, Protease Inhibitor, SYBR Green Assay, Mutagenesis, Purification, Gel Extraction, Imaging, Sequencing, Negative Control, Real-time Polymerase Chain Reaction, Software, Expressing