Function of the Plant DNA Polymerase Epsilon in Replicative Stress Sensing, a Genetic Analysis.
Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis (Arabidopsis thaliana). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote.
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
José-Antonio
JA
Pedroza-García
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
Christelle
C
Mazubert
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
Ivan
0000-0002-5169-4335
I
Del Olmo
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
Mickael
M
Bourge
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
Séverine
S
Domenichini
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
Rémi
R
Bounon
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
Zakia
Z
Tariq
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
Etienne
E
Delannoy
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
Manuel
0000-0002-4640-6511
M
Piñeiro
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
José A
0000-0002-2963-7641
JA
Jarillo
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
Catherine
C
Bergounioux
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.).|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.).
Moussa
M
Benhamed
Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Évry, Université Paris-Saclay, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.); cecile.raynaud@u-psud.fr.|Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France (J.A.P.-G., C.M., S.D., R.B., Z.T., E.D., C.B., M.B., C.R.); cecile.raynaud@u-psud.fr.|Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM 28223-Pozuelo de Alarcón (Madrid), Spain (I.d.O., M.P., J.A.J.); and cecile.raynaud@u-psud.fr.|Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France (M.B.) cecile.raynaud@u-psud.fr.
Cécile
0000-0002-5231-8120
C
Raynaud
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics; D004257:DNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D018506:Gene Expression Regulation, Plant; D063990:Gene Ontology; D006918:Hydroxyurea / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D030821:Plants, Genetically Modified; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
D029681:Arabidopsis Proteins; D018744:DNA, Plant; D019384:Nucleic Acid Synthesis Inhibitors; D004257:DNA Polymerase II; D006918:Hydroxyurea