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A functional approach reveals a genetic and physical interaction between ribonucleotide reductase and CHK1 in mammalian cells.

Taricani L, Shanahan F, Malinao MC, Beaumont M, Parry D - PLoS ONE (2014)

Bottom Line: Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes.Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts.These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.

View Article: PubMed Central - PubMed

Affiliation: Merck Research Laboratories, Palo Alto, California, United States of America.

ABSTRACT
Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.

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Quantitation of dNTPs and γ-H2AX phosphorylation in U20S cells following depletion of RRM1 and RRM2 subunits of Ribonucleotide reductase.Cells were transfected with RRM1, RRM2 and Luciferase control (untreated or treated with 1 µM GEM or 1 µM CAFdA for the last 2 h) before harvesting at 30 h. At 30 h after siRNA transfections, (a) NTP/dNTP extractions were prepared and quantified. (b) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. Data performed in triplicates. Error bars represent standard deviations (SD) between experiments.
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pone-0111714-g001: Quantitation of dNTPs and γ-H2AX phosphorylation in U20S cells following depletion of RRM1 and RRM2 subunits of Ribonucleotide reductase.Cells were transfected with RRM1, RRM2 and Luciferase control (untreated or treated with 1 µM GEM or 1 µM CAFdA for the last 2 h) before harvesting at 30 h. At 30 h after siRNA transfections, (a) NTP/dNTP extractions were prepared and quantified. (b) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. Data performed in triplicates. Error bars represent standard deviations (SD) between experiments.

Mentions: Ribonucleotide reductase is a target of hydroxyurea (HU), gemcitabine (GEM), and clofarabine (CAFdA). HU, GEM, and CAFdA are RNR inhibitors, which deplete dNTP pools to inhibit DNA replication [30]–[34]. Subsequent stalled replication or DNA damage induces phosphorylation of Chk1 S345 and γ-H2AX S139 in S phase checkpoint [18], [20], [35]. To evaluate effects on dNTP pools, we compared the depletion of the two RNR subunits, RRM1 and RRM2 with GEM and CAFdA, known inhibitors of RNR. Studies have shown that GEM and CAFdA treatment suppress dCTP and dGTP & dATP pools but do not affect the dTTP pools [32]–[34]. In control cells, transfected with siRNAs directed against luciferase, dGTP was the smallest dNTP pool (0.08±0.05), compared with dCTP (0.13±0.01), dATP (0.43±0.13), dTTP (0.73±0.04) (Figure 1A; Table 1). Ablation of RRM1 or treatment of cells with GEM and CAFdA, known inhibitors of RNR resulted in depletion of dATP, dCTP, dGTP pools but did not affect the dTTP pools (Figure 1A; Table 1). Ablation of RRM2 produced similar outcomes, with the exception of dCTP levels, which remained unchanged when compared to control cells. Thus, specific depletion of RRM1 and RRM2 leads to diminished dNTP pools.


A functional approach reveals a genetic and physical interaction between ribonucleotide reductase and CHK1 in mammalian cells.

Taricani L, Shanahan F, Malinao MC, Beaumont M, Parry D - PLoS ONE (2014)

Quantitation of dNTPs and γ-H2AX phosphorylation in U20S cells following depletion of RRM1 and RRM2 subunits of Ribonucleotide reductase.Cells were transfected with RRM1, RRM2 and Luciferase control (untreated or treated with 1 µM GEM or 1 µM CAFdA for the last 2 h) before harvesting at 30 h. At 30 h after siRNA transfections, (a) NTP/dNTP extractions were prepared and quantified. (b) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. Data performed in triplicates. Error bars represent standard deviations (SD) between experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222937&req=5

pone-0111714-g001: Quantitation of dNTPs and γ-H2AX phosphorylation in U20S cells following depletion of RRM1 and RRM2 subunits of Ribonucleotide reductase.Cells were transfected with RRM1, RRM2 and Luciferase control (untreated or treated with 1 µM GEM or 1 µM CAFdA for the last 2 h) before harvesting at 30 h. At 30 h after siRNA transfections, (a) NTP/dNTP extractions were prepared and quantified. (b) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. Data performed in triplicates. Error bars represent standard deviations (SD) between experiments.
Mentions: Ribonucleotide reductase is a target of hydroxyurea (HU), gemcitabine (GEM), and clofarabine (CAFdA). HU, GEM, and CAFdA are RNR inhibitors, which deplete dNTP pools to inhibit DNA replication [30]–[34]. Subsequent stalled replication or DNA damage induces phosphorylation of Chk1 S345 and γ-H2AX S139 in S phase checkpoint [18], [20], [35]. To evaluate effects on dNTP pools, we compared the depletion of the two RNR subunits, RRM1 and RRM2 with GEM and CAFdA, known inhibitors of RNR. Studies have shown that GEM and CAFdA treatment suppress dCTP and dGTP & dATP pools but do not affect the dTTP pools [32]–[34]. In control cells, transfected with siRNAs directed against luciferase, dGTP was the smallest dNTP pool (0.08±0.05), compared with dCTP (0.13±0.01), dATP (0.43±0.13), dTTP (0.73±0.04) (Figure 1A; Table 1). Ablation of RRM1 or treatment of cells with GEM and CAFdA, known inhibitors of RNR resulted in depletion of dATP, dCTP, dGTP pools but did not affect the dTTP pools (Figure 1A; Table 1). Ablation of RRM2 produced similar outcomes, with the exception of dCTP levels, which remained unchanged when compared to control cells. Thus, specific depletion of RRM1 and RRM2 leads to diminished dNTP pools.

Bottom Line: Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes.Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts.These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.

View Article: PubMed Central - PubMed

Affiliation: Merck Research Laboratories, Palo Alto, California, United States of America.

ABSTRACT
Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.

Show MeSH
Related in: MedlinePlus