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

Lorena Taricani; Frances Shanahan; Maria-Christina Malinao; Maribel Beaumont; David Parry

doi:10.1371/journal.pone.0111714

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

PLoS One. 2014 Nov 6;9(11):e111714. doi: 10.1371/journal.pone.0111714. eCollection 2014.

Authors

Lorena Taricani¹, Frances Shanahan¹, Maria-Christina Malinao¹, Maribel Beaumont¹, David Parry¹

Affiliation

¹ 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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Line, Tumor
Checkpoint Kinase 1
DNA Damage
DNA Replication*
Deoxyribonucleotides / metabolism
Histones / metabolism*
Humans
Phosphorylation
Protein Kinases / metabolism*
RNA, Small Interfering / metabolism
Ribonucleoside Diphosphate Reductase / antagonists & inhibitors*
Ribonucleoside Diphosphate Reductase / genetics
Ribonucleotides / metabolism
Tumor Suppressor Proteins / antagonists & inhibitors*
Tumor Suppressor Proteins / genetics

Substances

Deoxyribonucleotides
H2AX protein, human
Histones
RNA, Small Interfering
Ribonucleotides
Tumor Suppressor Proteins
ribonucleotide reductase M2
RRM1 protein, human
Ribonucleoside Diphosphate Reductase
Protein Kinases
CHEK1 protein, human
Checkpoint Kinase 1

Grants and funding

Funding for this work was provided entirely by Merck Research Laboratories. The funder (Merck Research Labs) provided support in the form of salaries for authors [LT, FS, MCM, MB and DP], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.