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.
Highlights
Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides, the essential precursors of DNA synthesis in all organisms
We present evidence that Chk1 activation is functionally and genetically linked to Ribonucleotide Reductase (RNR), a finding reinforced by the fact that these critical enzymes in DNA replication checkpoint physically interact in mammalian cells
These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation, providing new insights into understanding the mechanism of action for RNR and CHK1 in DNA replication and checkpoint
Summary
Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides, the essential precursors of DNA synthesis in all organisms. RNR is an important enzyme in the early stages of DNA synthesis responsible for maintaining a balanced supply of dNTPs required for DNA synthesis and repair. RNR plays an important role in genetic fidelity AND cell viability [1,2]. Failure to control the dNTP levels leads to cell death and genetic abnormalities [3,4]. The large subunit RRM1 contains the catalytic site, the substrate-specifity site, and the activity site [2]. The RRM2 subunit contains an iron center generated tyrosyl free radical that can be scavenged by hydroxyurea [6]. Like RRM2, p53R2 can substitute for RRM2 to form an active enzyme with RRM1 [8]
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