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

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.

Figure 1. 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.

Figure 2. Depletion of RRM1 and RRM2 subunits of Ribonucleotide reductase inhibits DNA synthesis and induces phosphorylation of Chk1 and γ-H2AX in U2OS cells.

(a) Following 30 h siRNA transfections, cells transfected with RRM1, RRM2 and Luciferase control (untreated or treated with 1 mM HU for the last 8 h) were stained with BrdU and PI and examined by flow cytometry. (b) Extracts were immunoblotted as indicated. (c) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. ± denotes SD. (d) Cell proliferation was assessed with clonogenicity assay. Cell proliferation was expressed as a percentage of luciferase control cells. Results are expressed as the percentage of colony-forming efficiency and are the means of at least three independent experiments.

Figure 3. RRM1/Chk1 and RRM2/Chk1 co-depletion enhances the accumulation of DNA damage, apoptotic response and effects cell proliferation in U2OS cells.

Cells were transfected with Chk1, RRM1, RRM2, RRM1/Chk1, RRM2/Chk1, and Luciferase control (untreated or treated with 1 mM HU for the last 8 h) before harvesting at 30 h. At 30 h after siRNA transfections, (a) extracts were prepared and immunoblotted with the indicated antibodies. (b) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. (c) Cells were collected at indicated time points indicated and analyzed for activated caspases. Data performed in duplicates. Error bars represent SD between experiments. (d) Cell proliferation was assessed with clonogenicity assay.

Figure 4. RRM1 and RRM2 subunits of Ribonucleotide reductase interact with Chk1 and Polα.

(a) At 30 h after siRNA transfections of the luciferase, RRM1, and RRM2 siRNA duplexes, extracts were prepared for immunoprecipitations. Cells transfected with Luciferase were incubated with 1 mM HU for 30 min. Chk1 was immunoprecipitated from luciferase (positive control), Luciferase + HU, RRM1, and RRM2 depleted cells with Chk1 antibodies (MAb58D7) cross-linked to protein A and were immunoblotted as indicated. Chk1 immunoprecipitations were peptide blocked as a negative control. (b) Polα was immunoprecipitated from luciferase (positive control), luciferase +HU, RRM1, and RRM2 depleted cells depleted cells with Polα antibody (SJK132-20) cross-linked to protein G and Western blots were immunoblotted as indicated. (c) Whole cell extracts were immunoblotted as indicated.