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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 Nov 6;9(11):e111714.

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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Significance Statement: 

Ribonucleotide reductase (RNR) is a therapeutically relevant oncology drug target.  Activation of CHK1 is a key component of the replication checkpoint induced by RNR inhibitors.  Ablation of CHK1 function in this context leads to replication checkpoint override, accumulation of DNA damage and cell death.  Despite many studies, the role of RNR in replication checkpoint and Chk1 activation remained unclear.  Taricani et al. investigated the relationship between DNA replication checkpoint and Ribonucleotide Reductase and revealed a functional and physical interaction between RNR and CHK1.  In this manuscript, they used established mechanistic markers of checkpoint activation and DNA damage (ie. g-H2AX, RPA32 S33, CHK2 T68, CHK1 S345) to not only complement existing genetic data, but also expand our understanding of the CHK1/RNR relationship in mammalian cells.  Furthermore, their data supporting the physical interaction between CHK1 and RNR is entirely novel, thus reinforcing the functional and genetic relationship between the two components.  These observations provide a mechanistic rationale for combination strategies using anticancer drugs, such as gemcitabine and cytarabine, together with Chk1 inhibitors.  Furthermore, these data suggest that selective RRM1 or RRM2 compounds could have therapeutic utility, both as a monotherapy and in combination with CHK1 inhibitors or other chemotherapy for drug development.

Figure Legend:

g-H2AX (DNA damage) assessment following RRM1, RRM2, CHK2 siRNA-mediated knockdowns in combination with Chk1 inhibition using siRNA or selective Chk1 inhibitor, SCH900776 (MK-8776) for 2h in U2OS cells. siRNA-mediated knockdown of Chk1 with 1mM hydroxyurea (HU) overnight was used as a positive control.

A Functional Approach Reveals a Genetic and Physical Interaction between Ribonucleotide Reductase and CHK1 in Mammalian Cells [Public]

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