A functional approach reveals a genetic and physical interaction between ribonucleotide reductase and CHK1 in mammalian cells.
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.
Merck Research Laboratories, Palo Alto, California, United States of America.
Lorena
L
Taricani
Merck Research Laboratories, Palo Alto, California, United States of America.
Frances
F
Shanahan
Merck Research Laboratories, Palo Alto, California, United States of America.
Maria-Christina
MC
Malinao
Merck Research Laboratories, Palo Alto, California, United States of America.
Maribel
M
Beaumont
Merck Research Laboratories, Palo Alto, California, United States of America.
David
D
Parry
D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004261:DNA Replication; D003854:Deoxyribonucleotides / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D012262:Ribonucleoside Diphosphate Reductase / Q000037:antagonists & inhibitors* / Q000235:genetics; D012265:Ribonucleotides / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics
D003854:Deoxyribonucleotides; C468783:H2AX protein, human; D006657:Histones; D034741:RNA, Small Interfering; D012265:Ribonucleotides; D025521:Tumor Suppressor Proteins; C093713:ribonucleotide reductase M2; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1